[ijrocHEMJ

BIOCHEMISTRY
META BOLI SM
Three irreversible steps of glycolysis are Glucokinase, PFK-1 and pyruvate kinase
Total and net ATP produced in aerobic glycolysis are.9.&. 4 respectively
Rate limiting enzyme of glycolysis is PFK 1 (committed enzyme)
Positive allosteric regulators of PFK-1 are: fructose 2,6 bisphosphate, AMP
Negative allosteric regulators of PFK-1 are ATP, citrate, protons

PASTEUR EFFECT
Inhibitory effect of 02 on glycolysis is called as Pasteur effect.
It is due to decreased AMP/ATP ratio. AMP has positive effect on PFK-1 and so
decreased level of AMP causes inhibition of glycolysis

CRABTREE EFFECT
Relative anaerobiosis produced when glucose concentration is increased in constant
. supply of oxygen. (lactic acidosis - inborn error of metabolism)

PDH complex (Q)

In mitochondria converting pyruvate (3C) to acetyl co A (2C)
Oxidative decarbocxylation - releasing NADH
Enzymes of PDH Complex are
1. PDH Pyruvate dehydrogenase
2. Dihydro-lipoyl transacetylase
3. Dihydro-lipoyl dehydrogenase
Co enzymes :
1. Thiamine (81)
2. Lipoic acid (sulphur containing)
3. Co A SH (85)
4. FAD (82)
5. ·NAD (83)
Therefore 8 complex supplied with dextrose fo r its metabolism

TC A CYCLE : KEY POIN TS

IRREVERSIBLE EN ZVMES of TCA cycle
- Citrate Synthase
- Alpha keto glutarate dehydrogenase complex
Total/Net ATP produced from one acetyl Co A 10/10

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T CA CYCt.E: A l.t.OSTE RIC EN ZY MES (Q) & THEI R RE&VLHO RS

Enzyme Positive allosteric regulators N egative regulator

Citrate synthase · •· ATP (reduce rot e. of cycle)
NADH
Succinyl Co A
Fatty acyl CoA (Q)
(also required for synthesis of
fatty acids)
Isocitrate ·•• ATP
dehydrogenase NADH
Alpha keto glutarate ATP
dehydrogenase GTP
NADH
Succin I Co A
GLU CONEOGENSIS
In liver, kidney, GIT
Regulation of &luconeogenesis

Following enzymes are regulatory

1. Pyruvote carboxylase
2. PEP carboxykinose
3. Fructose 1,6 bisphosphatase (e1>unter enzyme of PFK 1)
4. Glucose 6 phosphatase

HORMON AL CON TROL

Gen~ expl"tssion : !nsulfn t--epl'CSs Pyruvatc Corboxylase (therefore in diabetes

increases glucose as inst.11in represses carboxylase which supresses pyruvate
carboxylase from gluconeogenesis )
GLVCAGON forms cAMP. Via cAMP: GIUCQgOn induces gene of PEP carboxykinase ,F
1, 6 bisphosphatase .glucose -6- phosphatase
Glucagon via cAMP: &lucagon iMctivatcs Pyrwate. Kinase (used in glycolysis)

AUosteric control:
&;styl CoA: Activator of Pyruvate Corboxylose
Source of occtyl CoA fram fatty acid beta oxidation (child with
middle chain ocyl CoA de.ficiency will have. frequent
hlf?~lyCQetnic: attack due to absence of this.}
_ _ _ !!s,ulator of PEP Ca~kil'\an
@, C;ttrsrt,:
AMI F2,6
1Activatorofof Fructosel1,66
, tnhi~'tor Fructose
bisehose_hotasc
bisph_oSD,_h,.,_citas.
....__ _ _ _ _ _ __.

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PFKl positive regulators act as negative regulator of gluconeogenesis : contra-

regulation

GLYCOGEN METABOLISM
GLYCOGENESIS

Requirements
- Insulin
- Glucose [UDP Glucose - nucleotide]
- LIVER
Rate limiting enzyme: Glycogen synthase[active in dephosphorylated form]
Bronchi ng enzyme: Amy lo 1. 4-1. 6 glucan transferase/transglucosidase

GL Y COGENOL Y SIS

Glycogen phosphorylase

- Rate limiting enzyme 40

- Active in phosphorylated form Exogenous

- Needs PLP[Vitamin B6] c:,1 30

C

:ii
0
Needs Pi
Cleaves 1-4 bond .= 1.4 glucosidase 20

- Release glucose in glucose 1 9:

phosphate 8
:,
10 Glycogen Gl11con,-090tiM1S

Glucose as a fuel during fasting '&

and starvation 4 2
Carbohydrate metabolism during Days
fasting starving time
- Dietary glucose last for 1.5 to 2 hours
- Gluconeogenesis: Begins at 2-3 hrs. Peak at 2-3 days. Decline at 7 day. Last for
life (no glucose RBC do hemolysis)
Glycogenolysis : begins at 2 - 3hrs. last for 18-24hrs

LIPID AN 'D PROTEIN METABOLISM

.,.

Fatty acid mobilization starts at 12 hours (From triacyl glycerol from adipose cells ,
major organ is :liver )
Ketone body appear in body fluids by 48 - 72 hours
Muscle protein breakdown occurs considerably by 5 days and beyond

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Phases of glucose homeostasis

Phase 1 Phase 2 Phase 3 Phase 4

Nut ritional Well fed Post-Absorptive Fasting Prolonged

stat us state state fasting/
Starvation

Source of Diet Hepatic glycogen Hepatic and Renal and

glucose and Renal hepatic
Gluconeogenesis gluconeogenesis gluconeogenesis

Tissues All All tissues, but in Brain and RBCs Brain at a slower
using liver, muscle and an~ cells lacking rat e, RBCs
glucose adipose tissue, the mitochondria; normal rate
rate of utilization is small amount by
slowed. muscle.
---
M,ajorfuel Glucose Glucose Glucose and Ketone bodies
of brain ketone bodies and glucose

LI PI DS
CARNITINE SHUTTLE
On mitochondrial membrane (liver)
Enzymes of OMM (outer mitochondria): Acyl Co A synthetase & CPT-I
Enzymes of IMM : carnitine/ acyl carnitine translocase & CPT-I
Malonyl Co A is the blocker of CPT-I
Carnitine is beta hydroxyl gamma trimethyl ammonium butyrate (Q)

fATTY ACID BETA O XI DATI ON

Mitochondrial matrix proc~s

Cyclical reaction with the set of four enzymes in each cycle
Purpose is ATP Production
Accident is ketogenesis on excess of it in liver
Unsaturated acyl chain produces lesser ATP compare to saturated fatty acid chain of
equal length
4 Enzymes of each cycle of beta Oxidation
1. FAD li nked Acy I Co A Dehydrogenase (F ADH2)
2. Hydration by Enoyl Co A Hydratase

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3. NAD linked beta hydroxy acyl Co A Dehydrogenase (NADH2)

4. Thiolase
In palmitic acid - 7 cycles
In 1 cycle: 1 FADH2 + 1NADH2
16C palmatic acid produces : 108 ATP, 2 ATP spent in carnitine shuttle : 106 atp
JAMAICAN VOMITIN G SICKN ESS is caused by eating unripe fruit of the ' Akee
tree' , which contains the toxin HYPOGLYCIN which inhibits. MCA dehydrogenase
activity (inhibit beta oxidation)
Zellweger ' s ( cerebral hepatorenal syndrome) syndrome is either due to lack of
peroxisomes or the number of peroxisome may be normal with no enzymes (deposition
of very long chain fatty acids)
- If perioxysomal number is normal, peroxisomes are empty
Defective enzyme in Refsum ' s disease is "phytanic acid alpha oxidcise", (phytanic acid
alpha hydroxylase

FATTY LIVER
Deposition of triacyl glycerol in liver affect architecture and function of liver
Excess formation of FA, synthesis is normal with decreased secreti~n
Factors leading to fatty liver

Puromycin Protein deficiency

Ethionine EFA deficiency
CC14 Vitamin deficiency
Chloroform VLDL - needs lecithine, choline,
Phosphorous vitamins to form
Lead Alcohol (Excess)- decreased beta oxidation of
Arsenic fatty acids, increased NADH
Orotic acids
Lipotrophic factors: (help in mobilixzation of TAG from liver)

Choline Omega 3 fatty acid

Lecithine Selenium
Inositol Vit E
Methionine
Betain
[CLIMB]

UREA CYCLE
Rate limiting enzyme is CPS-1( Carbamoyl phosphate synthetase-1)
NAG(N Acet yl Glutamat e) is a posi t ive allosteric modifier of CPS -1 (GLUTAMATE
source of ammonia) (Q)

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Deficiency of OTC (ornithyine transcarbomylase) Enzyme is associated with Orotic

Aciduria (increase formation of carbomyl phosphate - increase in pyrimidine
intermidiates)
Urea cycle food disorder : urine product has lot of metabolic products of thiamine
Sources of nitrogen for urea is free NH3 from glutamate and whole of the Aspart ate
(aspartate - oxaloacet.gte ) (both NH3 is glutamate
ortant odour of urine in aminoaciduria
Cab ncid Bu
Ch ur/swea Isovaleric aciduria
Mo ty odour: Phenylketonuria (d/t phenyl acetate ) ( Q)
Bur r: · e Disease
Tom Cat Urine smell: lase deficienc
Swimmin ool odour:

Amino acids Special groups

Guanidium-d
.... Indole-B
Histidine Imidazole-13
Proline P rrolidine-a
Tyrosine Phenol-B
SECONDARY STRUCTURE OF PROTEIN

Alpha Helix:
- Most common and stable form
- Spiral structure
- 3.6 residue /turn
- Right handed
- Proline, hydroxyproline not found in the alpha helix
Beta Pleated Sheet
- May be parallel or anti parallel
- It is a structural motif of the silk fibroin, flavodoxin, carbonic anhydratase
- Beta bends are abrupt turns in the chain favoured when small AA like glycine or proline
are there
Enzymes helping the structural organization of collagen molecule
- Lysyl hydroxylase
- Prolyl hydroxylase
- Lysyl oxidase
- Need vit C and copper

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Lysyl & Prolyl hydroxylase :

Cofactors are ascorbic acid (vitamin C) and a- ketoglutarate
- Helps forming the collagen fibrill. (post translation modification of polypeptide,
converting lysine to hydroxyl lysine)
Lysyl Oxidase
- Cu dependent enzyme ,
- Oxidative deamination of E-amino group of lysine & hydroxylysine
- A Idol condensation & schiff' s base f6rmation
- Stabilises the staggered arrangement

RATE LIMITING ENZYMES OF VARIUOS PATHWAYS

Pathway Rate limiting enzyme - -

Glycolysis PFK-1
Gluconeogenesis Fructose 1,6bisphosphatase
Glycoqenesis Glycogen synthase
Glycogenolysis Glycogen phosphorylase
Fatty acid synthesis Acetyl Co A carboxylase
Cholesterol synthesis HMG Co A reductase
Bile acid synthesis 7 alpha hydroxylase
Vitamin t> activation 25 hydroxylase

List of enzymes active in phosphorylated and dephosphorylated forms

- -

~nzyme a_c!~E: _i_!'l_ dephosph~~_yJated form ___ J!Jzy_me ~~tive i_n_eb9~p_~oryla_!~d f 9,:~_ __ ;
- Glucokinase - Fructose 2,6 bisphosphatase
- PFK-2 ( 1 is allosteric enzyme) - Glycogen phosphorylase
- Pyruvate Kinase - Hormone sensitive lipase
Glycogen synthase - ATP Citrate Lyase
Acetyl Co A Carboxylase - Phosphorylase kinase
HMG Co A Reductase HMG Co A Reductase Kinase

Important metabolic disorders and associated enzyme deficiency

Disorder Enzyme deficiency

Essential pentosuria Xylitol dehydrogenase
Essential Fructosuria Fructokinase
Hereditary fructose intolerance Aldolase B
Classical galactosemia Galactose-1-phosphate uridyl transf erase
Benign galactosemia Galactokinase
Jamaican vomiting sickness Unripe fruit of the 'ake
Zellweger's Absence of peroxisomes
cerebrohepatorenal syndrome

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Ref sum's disease Phytanic acid a-oxidase/(Phytanic acid a-hydro-

xylase).
Tyrosinemia I Fumaryl acet oacetat e hydrolase
Tyrosinemia n Tyr osine aminotransferase
Alkai:,tonuria (Q) Homoqentisic Oxidase
Albinism Tyrosinase
Homocystinuria type I Cystathione beta synthase
Homocystinuria type n Homocysteine methyl transf erase
Homocvstinuria type m N5N10 Methylene reductase
Cystathionuria Cystathionase
Benign Histidenemia Histidase
MSUD [Maple Syrup Urine Disease] Branched chain alpha keto acid
dehydrogenase/ decarboxylase deficiency

GLYCEMIC INDEX

Defined as an "Effect of 50 gm of carbohydrate in a particular food on blood glucose

level as compared to 50 gm of anhydrous glucose".
·Ratio of incremental area under glucose tolerance curve after 50 gm of test meal to
that of the 50 gm of the glucose is called as glycemic index.
Glucose = 100

Glycemic index of various food items

A. Potato chips: 80-90
B. Bread: 70-79
C. White rice: 70-79
D. Brown rice: 60-69
E. Banana: 60-69
F. Legumes, peanuts: 35-40
G. Milk, icecream : 35-40

MCQS 0.4 ....

1. 7 y/o child presenting
with blistering and
0.3

hyperpigmentation 0.2
lesion of skin 0.1
Urine examination
shows sharp absorption 500 550 600 650
at wave length 400 nm.
SORET BAND
Seen in porphyria

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Hepatic Vs Erythropoietic porphyria (Based on organ most affected)

If liver enzymes affected it is hepatic porphyria

If bone affected it is erythropoeitc porphyria
Hepatic: Acute/Chronic
- Acute: AIP, HCP. VP
- Chronic: PCT
Erythropoetic : CEP, PP

ON E LIN ERS
ALA synthase is the rate limiting enzyme of hepatic biosynthesis of heme.
ALA synthase II deficiency result in X-linked sideroblastic anemia
In general, porphyrias are inherited in autosomal dominant manner, with the exception
of congenital erythropoietic porphyriav[CEP], which is inherited in th'e recessive mode.
Pure neurological porphyrias: AIP
Pure cutaneous porphyrias: PCT, CEP, PP
Both cutaneous and neurological porphyrias: HCP and VP

Isoele;ctric pH

AA have no net charge

No electrophoretic mobility
Isoelectric focusing uses this principle

Test for carbohydrates

1. Molisch test
2. Benedicts test
3. Barfoed test
4. Seliwanoff test
5. Osazone test

None Traces of Moderate Lo,.e

reducing sugar • mount of
reducin&
supr

Few drops of alpha naphthol in 5ml of benedicts solution + 8 drops of urine

acidic solution - molisch test Co ent : reducin su ar in urine

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\ .' '

Petalic structure Seliwanoff test Glucosazone/ frucatose -

Maltosazone Presence of fructose negative needle shaped crystals
for glucose
t
I

Powder puff - lactosazone

1st control
2nd : red dirty ppt
Presence reducin monosaccaride

Colour reaction of proteins

Ninhydrin test
Biuret test
Xanthoproteic test
Hopkin cole test
Pauly's test
Millon's test
Sakaguchi test
Sulfur test

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 Positive N •catlve Positive

NINHYDRIN TEST
- For alpha amino acid
- Ruhemann purple
- Staining of amino acid on BIURET TEST
chromatography paper Copper coordination complex with 2 peptide
- Except proline which has imino group bond ( AA with atleast 1 bond)
ives ellow colour

I I

II',!/. L... .
-------- - ll 'HOPKIN COLE TEST / ALDEHYDE TEST
XANTHOPROTEIC TEST I'\ I Indole r ing gets oxidized in presence of
Nitration of aromati~,rj ng gives Y.ellowicolou}, ,ner~uric: sulfate in .sulfuric acid, to which
Phenylamine, tyrosine cind't ryptophan : formaldehyde reacts to produce p~rple r ing
Positive for aromatic amino acid Rosi_tiv~ for trypto han
,-------------,

.A
I .

PAULY'S TEST MILLON' S TEST

Cherry red colour: histidine Mercuric sulfate in sulfuric acid(Millon's
Sulfanilic acid and sodium nitrite is used in reagent) is added on with sodium nitrate
alkaline medium Positive for tyrosine
Positive for histidine Dir red

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SULFUR TEST
~ 1a Lead acetate in alkaline medium
SAKAGUCHI TEST Cysteine and cystine give positive test
Molish reagent and bromine water Methionine does not give positive test due to
Arginine guanidium group Sul hur in _thioether linkage

PR OTEIN PRECIP I T ATION TEST

Heat coagulation test
· Heller's test
Half saturation test (globulin gets ppt)
Full saturation test (albumin gets ppt)
Iso-electric point ppt

Heat coagulation test of proteins

i No coagulum
formation

Proteins Absent Proteins Present

Negative Test Positive Test

None Traces of Moderate large
recludng sugar amount of
Heat .coagulation test reducing
su,ar

For reducing substance .. _benedicts stest

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ROTH ERA'STEST

Tests for Ketone bodies

Rothera' s Test Control for
comparison Positive test
Principle: Nitroprusside in alkaline medium
reacts with a ketone group to form a purple HAYS SULPHUR TEST
ring. It is given by acetone and acetoacetate, Test for Bile salts
but not by Beta hydroxy butyric acid. In the control , sulphur powder remains
immiscible with the underlying liquid.
In the positive test, the sulphur powder
sinks to the bottom.
Int~rpretatione Bile salts and bile pigments
are present in urine in
obstructive jaundice.
Tests for bile pigments
Fouchet's test
Procedure:
Take 5 ml of 10% BaCI, to 10 ml of urine and
filter . Dry the filter paper and add a few drops
of Fouchet's reagent (Prepared by adding 10
mg of 10% FeCl3 to 100 ml of 25% TCA). A
green color is obtained due to oxidation of
bilirubin to biliverdin.

MCQ
1) Which one of the following statements concerning glycolysis is correct?

a. The conversion of glucose to lactate requires the presence of oxygen.

b. Hexokinase is important in hepatic glucose metabolism only in the absorptive period
following consumption of a carbohydrate-containing meal.
c. Fructose 2,6-bisphosphate is a potent inhibitor of phosphofructokinase.
d. The regulated reactions are also the irreversible reactions.

Ans: d

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2) After a 72-hour fast the substance likely to the involved in the formation of energy would
be:

a. Muscle glycogen
b. Aminoacid
c. Liver glycogen
d. Acetoacetate

Ans: d (beta hydroxyl butyrate more ATP than acetoacetate)

3) Sorghum causes pellagra due to the presence of:

a. Lysine
b. Leucine
c. Tryptophan
d. Alanine

Ans: b (completes with tryptophan for absorption and hepatic entry therefore leading to
pellage, kyneurenin pathway is affected by kynureninase and quinoteinate PRT inhibition)

4) Which one of the following sequences places the lipoproteins in the order of most dense to
least dense?

a. HDL/VLDL/ chylomicrons/LDL
b. LDL/ chylomicrons/HDL/VLDL
c. HDL/LDL/VLDL/chylomicrons
d. VLDL/ chylomicrons/LDL/HDL

Ans: c (most dense is HDL, least dense is chylomicrons)

5) Why glucose is stored as glycogen

a. Has compact structure

b. Can supply glucose for one week of fasting
c. Has many reducing ends.
d. Can be stored in multiple organs

Ans : a

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6) Patient presents with pallor and cyanosis with

following Presentation shown in picture where
hyperpigmentation of knuckle is seen

a) Vitamin B12 deficiency

b) Folic acid deficiency
c) Iron deficiency
d) Vitamin B6 deficiency

ANS: hyperpigmentation of knucles seen in : PCOS, Addison's disease an vit B12 deficiency (a)

7) Enzymes affected in vitamin B12 deficiency

A. Methyl malonyl CoA mutase/isomerase

B. Methionine synthase/ homocysteine methyl tansferase
C. Folate reductase
D. Homocysteine synthase

Ans : a,b

8) Which of the following statement is true regarding "Cyanide"

a. Only minimally inhibits the ETC because cytochrome oxidase is the terminal component of
the chain
b. Inhibits mitochondrial respiration but energy production is unaffected
c. Also binds the copper of cytochrome oxidase
d. Bind to Fe+++ of cytochrome a3

Ans : affects ETC and targets cytochrome IV, cytochrome oxidase has heme with iron and
copper, ferric to ferrous and cyanide blocks it) (d)

9) Cerebrohepatorenal syndrome. Which of the following substance accumulates in brain?

a. Pyruvate
b. Very long chain FA
c. Short chain FA
d. Glycogen

Ans: B

10) Regarding proteoglycans, which of the following statement is wrong?

a. Chondroit in sulphate is a proteoglycans

b. They hold less water
c. They are made up of sugar and amino acids
d. They carry charge

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Ans: mucopolysaccharide (complex carbohydrates)+ protein for complex proteoglycans, a,b,c

11) Which among the following reactions requires pyridoxal phosphate as a coenzyme?

a. Carboxylation
b. One carbon transfer
c. Glycogen synthesis
d. PDH complex

ans: c (+ glycogen phosphorylase) (carboxylation needs B7)

12) A 12 year old girl presents with history of one episode of

generalised fits and repeated episodes of red scaly skin rashes
for past 2 year. She was born at full term with normal milestones
to a non consanguineous parents. She also has history of psychotic
behaviour and difficulty in walking for past six months.

Urinary investigation by chromatography revealed presence of

multiple neutral amino acid and presence of Indican in the urine.

Which test is done in the urine to find out Indican in the urine

a) FeCl3 test

b) Guthrie test

c) Benedicts test

d) Obermeyer test

Ans: hartnupp's disease ( 20 AA - 2 basic, 3 acid), cant absorp tryptophan therefore pellagra
like features (d) - naphthol reacts with indacan blue ppt

13) A 17 year old male is admitted to medicine department with history of bizzare behaviour,
episodes of crying and screaming. On examination he had tremors in hand, incoordinated limb
movements, dysarthria and dysphagia was
observed.

On visual examination his eyes showed golden

brown ring around corneal rim which was
confirmed as KF ring[ Kayser Fleischer ring] on
slit lamp examination.

Following were biochemical findings: Serum ceruloplamin: 12 mg/di( normal level is 18-35
mg/di) Urinary copper(24 hr collection): 270 µg(normal is 20-50 µg)

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Copper content in liver (biopsy): 300 µgig of tissue (normal level is 20-50 pg/g)

Deficiency of which protein is responsible for such features

a) copper transporting ATPase 7 A

b) HGPRTase
c) copper transpo~ting ATPase 78
d) Sphingomyelinase

Ans : (c) wilsons disease, copper deposit in desmonts membrane , seen in minke's disease and
aceruloplasmia also (a is seen in menke kinky steely hair syndrome, help in transport of copper
from GIT to blood)

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GENETICS

1. HGPRTASEenzymedeficiencyleadsto Lesch Nyhan syndrome.

H)1M>xanthine guanine
pho1phoriboayl tranmrase
Guanine GMP
7 ~ ·
PRPP PPI

r •
Hypoxanthine guanine
phosphoribosyl tronsferoH
Hypoxanthlne IMP
PRPP PPI

Hypoxanthine guanine phosphoribosyl transferance

Self mutilation behaviour - finger eating
Accumulation of guanine and hypoxanthine - forms uric acid
Hyper-urecemia

2.UMP Synthase deficiency leads to hereditary erotic aciduria

I
Oiriydro-orolk:

l
Or011c aeill

ll=p~s)1
tronsfcrasc

-~1~
OMP Type I
(OIOllle monophosplllle orollc
oaduna

l ype2
orotlc
aciduria

UMP
(urid Int monop/1011 p/lale)

Orotic aciduria + urea cycle disorder = OTC deficiency

UMP synthesa > OTC deficiency

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3.Werner syndrome, Bloom syndrome and Rothmund

-thomson are caused by defective HELICASE enzyme
Disorder Defective Protein r Mutation Inheritance

Werner syndrome helic-ase WRN autosomal

-- recessive ,__
Bloom syndrome hellcases
JBLM autosomal
recessive

Rothmund - helicases RECQL4 autosomal

thomson recessive

1st enzyme in DNA repl ication - helicase (unwindase)

r efective Telomerase enzyme causes ID'SKERATOSIS CON GEN ITA

DYSKERATOSIS CONGENITA -of signs:

a) dystrophic nails,
b) patchy skin hyperplgmentation, and
c) ora l leukoplakia

Telomerase required for end replication of DNA

Telomerase is reverse transcriptase, RNA dependent DNA polymerases
TTAGGG hexanucleotide repeats
Increase telomerase in cancer
Normal act ivity in germ cells/ HSC, stem cells
Normal/ decreased activity seen in somatic cells

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5. Hereditary nonpolyposis colon cancer anaXeroderma pigmentosum

are caused by MMR-Mismatch Repair and NER-Nucleotide Excision
Repair repa ir defects ,respectively.

HNPCC - MMR defect

NER defect results in - trichohiodystrophy, Cockayae syndrome also
Xeroderma pigmentosa : accumulates thiamine diamers (bulky adults)

6 . SCID IS CAUSED BY DEFECTIVE NHEJ DNA REPAIR

Defective N onhomologous !nd Joining ..,_. (NH!J)
SeYerecombined lnvnunodeficiencydisease (SCIO)
Radlatlon.senshlve seYm combJMdiimmunodelicienc:ydlwase
(~SCIO)

CNfedlft HomologouJ Rtpelr(HRJ

AT-like dlsordtr (ATlO)
NIJmegen btH!cage JYnd~~ (NBS)
oom syn rome J
Wemet syndrome (WS)
Aothmond-Thornson ndrome (RTSl
Breast cancer suspectibility 1 and 2 (BRCA 1, BRCAl)

Dtfectlw OHA Nucl.odcle Exlmfon air (NER)

Xeroderma ptgmentosu,n,(XP)
Cockayne syndrome (CS)
Trlchothlodystrophy {TTD)
DN A IIM Elccldon Rtp1lr (IERt
MUTYli·llSsc&tfd polyposis (MAP)

DefectJv. ON A Mllrnatch Repair (MMR)

Hetedhary nonpolyposb colorect.al_cancer
__ (HNPCCl
_ _ _ _ _ _ __J

NHEJ seen with double strand base repair (DSB) (seen with crisper crasp 9)
BER also causes breast cancer
HNPCC = LYNCH syndrome

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7 . XERODERMA PIGMENTOSA IS AN EXAMPLE OF SPLICE SITE MUTATION

:
lntton [aon) t'.aon)
:,
l.Jlon1 lntton
DN A C ··::: I$~ } : "='::! ) t ;!! :::::' ::: ##!¢ : Q!

Primary
RNA e
bon ,
~
- ~~-· l
.....
u on)
) Exon)

transcrtpt

Altered mRNA
ee
h oo 1
51
tnt1on

1¥1#
l - EMlll Eaon ,
;:,
Primary introns (not required) is to be removed
If the intron becomes a part of exon it's a splice site mutation
Splicing done by : SNRNA
Post transcriptional modifications : capping, splicing and tailing {RNA changes)

1.Following is the sequence of enzymes in DNA replication

e prottlM
Sl11Ql...t'9nd binding
IIIIIIIIIU Ille
E) TM INdlng . . . . 19
sytt!Mllad ~
unwound . . . . . DNA. 1nu.a·- a·.,..._~
DNApol,-.ao.
DNA polymerase

en. llgglng snnd ..

Prllllase •Yllllletllles I st.ort
RNA p,lffllr, WIiiet\ t•
...,_.IIVDNA~
tofoml111011amt .........

Overall dlracUon o f ~

DNA primer required for PCR

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DNA Polymerase can act only from 5' -3' direction

5'- 3' exonnuclease activity (only prokaryotic DN A polymerase 1)
3'- 5' exonuclease - proof reading (all DNA)

2.Following is the sequence of steps of PCR

-....-
~·
3nl cycle :,OU, i,yc;fi,
1st cycle 2nd c:ycii,

.,..,..
-=-•
f
........

• - - l ..-
f .,..,..

- - · · ·~=
-
'-- ........
t"fldo CIIP7I

l - f
_..
.,..,..
' ---
-
.r.-
2"' • 10"coples

---
•
M·M*W+ W:Mil:\· Mhii:Ph
C:

l -~
= 400t)IH
.,..,..
........
M

2'=8coplH
2' = 2 copies

Denaturat ion 95C

Annealing - 55-65C
Extension 72C
RT-PCR - done if starting substrate is MRA - cDNA - dsDNA - PCR
Real time PCR = qPCR = dye used cyber green, bind double stranded DNA

3.Sequence of enzymes involved in DNA repair mechanism

..... ~c,.QR_,•
.. ,..
_,..
o•
... ----w--------1~;;;;..---- - ~ . ; __ _ • • lladalon - - by
:a• ,,.._vnth . .111

.. ..~· - -- -- - - - --
1. , ... • •
----- - - -~·
- - - - - - ~ \. . .. . loft

,.. ---------------- r,·

"· a· - - - -- - - - - - -- - - -- ~• Ml....... 1ch_.,_,

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Endonuclease - DNA polymerase - DNA ligase

4. Steps in Southern Blot

I

....
DN A CJCJCJCJ
fragmenL<s

er towel
Ion fll te
osegel
DN A &.:.;.~..:.I@ L:;;:.!::::::.!:!!;=!J .,_______...,.IIIJ~onge
sample Gel electrophoresis
uffcr
,..-c==c=====::;,..... lution

.
,..'""-:..-:..~-:.-:.~-:..-;-- "'..a J

_,
-'
Autoradtogr.aphy Comp emen ry Replica of DNA
pairing of probe Addition of on nylon filler
witb target DN A radioactive robe

For DNA - southern blotting , cDNA probe

Northern for - RNA, cDNA probe
Western - protein, need Ab and cDNA
South western blotting - DNA that binds to protein

5. Sequence of steps in Recombinant DNA technology

- ·- ·--- 1~ h
~~:~e
Human cell Sticky Recombinant Gene for
end DNA human
EcoRI
Gene for human 'DNA
V
endu
growth hormone recombination

Sticky__. \

- 0 --~
Bacterial cell DNA
EcoRI .. lnsertlo.::._
Bacterial ~
chromosome ~ ·
Bacterial cell for Q
Plasmld - - 9 containing gene for
human growth hormone

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Mc vector : plasmid

Sequence of I ipoproteins from anode to cathode on electrophoretic separation

Lipoprotein Electrophoresis Pattern

-ve
I I +ve

Origin Beta Pre-beta Alpha

Chylomlcrons - -

At beta - VLDL,pre beta= LDL, alpha - HDL

HDL - highest content - fastest movement
Chylomicron at site of origin

LIPOP ROTEINS

· Lipoprotein maximum phospholipid content - HDL

Max cholesterol - LDL
Exogenous TAG& unique APO B48 - chylomicron
Fat free carbohydrate r ich diet - VLDL increase in serum

7. Sequence of proteins involved in RNA interference

E PHtM16Qii
I
RN A Polymsuo II

'
f
- (A)A,

p,1-mif'lN A

-
As.,.m,netrlc
u..-iding
and -
wand sa6ec1lon
T-
fflll\NA dllpfe)I

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Pasha - cleave of cap and t ail

Dicer - cleave loop head (cytosolic enzyme)
RISC - causes mRNA arrest - complementary to mRNA (RNA scliencing complex)
binds on 3' end
Normal gene regulation

a. Arrangement of RNA according to their size &

concentration

--
rypo F-or rn f-unc lion Amount

r he a uucturAJ par,:
rR N A o r the rlbO•ome

W o • k• . . . brk:JQO
tRN A 1 1 11 ~'TTTT bO two a n m nNA one, - 1d%
.-, n, ln-u o.L. •cb.
,I ~ .....

mRN A t: nc OCJ •• ,o,. - 2 b'"1

p-l"O t•tn•

Most abundant rRN A. synthe.si z( d in nucltolus

c.ompartna of RNA
mRN A rRNA tRN A
m olecul•r 25,000 to
weight up to 1,000,000 23,000 to 30,000
, .000,000
(grams/mole)
shows base no obvious base no obvious base
base relattonshtp wllh retauonsh1p wi th DNA r ela tionship with ONA
relatfonshlp ONA because It Is because It Is because It Is
with DN A formed from the synthesized from only synthesized from onty
ONA template small sections of DNA small sectJons of DNA

Max molecular weight - rRNA

mRNA directly

SECTION 3
1 . PROOF READING IS THE ONLY PROCESS WHICH TAKES PLACE IN 3 ' - 5 '
DI RECTION

DNA polymerase - exonuclease activity

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j2. Kienow fragment lacks 5"-1Exmmdea1edirect1cml

K ienow fragment
DN A palymocaao I
Haloenzyae
Pmbtollf&i& ...- Large frag1nent

polyme.ue
do main
L
- -~ -:::: -~

5' ->
exora.,c!lcasc.,
--~ r

•
(Kienow lrQillllentJ

5' ·> 3' e,coructea;e

3' -> 5" eoconuclMse domain domain

· Kienow fragment : large f ragment (604 A A)

of b N A po,t :C. having DN A polymerlza'tion
and 3 ' --. 5 ' exonucleose activit ies, and is
widely used in molecular biology.
DNAP I-
Polymerase activity 5' -3'
Kienow fragment doesn't have 5'-3' exonuclease

ls-BN ApolymenlecomperedtoDN ApolymenteQllnotworkln~ dhectlan

Transcription proceeds in the 5'- 3' direction until it encounters a

terminator sequence that disrupts the protein-nucleic acid complex
and halts RNA synthesis

Has only 5'-3' polymerase activity

avr&-UN TBAN SLATEPREGIQN endofmRN Aisbindingstteaf

miRNA(for RNAi) ,
locationofSECIS (for selenocysteine synthesis) ,
binding site of JRP on IRE ofTfR. mllN
poly-A ~

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5 . VITAMIN C AN D KARE INVOLVED IN POST TRAN SLATION AL

MODIFICATION
Vitamin C - prolyl hydroxylase
Vitamin K dependent carboxylase, factor 2,7,9,10 to activated form

6. All except Ht_histone, is part ofhistone octamer.

l
Unkor
DNA

l
Nucleosome -146 BP DNA+ histone octamer

7 . POST TRAN SCRIPTION AL MODIFICATION MISSIN G ON

MITOCHONDRIAL MRN A S SPLICING
No introns
13proteins from 15659 BP in mitochondrial DNA
Only 13 proteins come from mitochondrial DNA rest from nuclear DNA
Only maternal inheritance
Doesn't follow Mendelian law

28SrRNA h I transfe 1

enzyme activity
and
is i · · y Riclp toxin from cast
Present in 23srRNA

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2. eEF-2 required for ribosomal translocation is inhibited

by Diphtheria Toxin via ADP ribosylation
11>1 A comoo,,11n1 en, - AOP•rlboeyun ,on
of a G p,oc11tn lhal c o - acavatlon ot
ndc,n-,t,1cyctasc ~ i n o Oproor,,ntn
(n) 8
_.,.le
co,npon«lt ol to111n ana- to
rec.pi.-. on cal ..,.mew-:
,...., .ac,....,· - A ioo,npol'IOI'• ponott·a Jatt mombtnnu.. .P toama membfatlO
of in,,.,_...,,_, e&II

.. __ -

HOO, ( a-
ca,.,..
(d) Du,16-up ol c.AMP
-•-•11•IMOCfrQfv><t•to • .._o_)
pout °"' ol .,,.

(e) A<N etoon aummary:

ATP
N AO Looad O p,oi.ln
(c) Adonytyt cycJA- """""" 1t1o
_ , ol ATP to cAMP. b

Diarrhoea mechanism

3. lncllsiran is the drug used for management of hypercholesterolemia

,based on RNA Interference technology, binds and inhibit PCSK9 mRNA
•
elaHAIUSC, ~ I•
---~r- - --
- ...... ~ - .... -"'N A

·---- ...... .I
- -·--
~- --
ausc.._a..

! ,,__..,

............. -- ,_,

PCSK9 leads to internalisation of LDL receptors , serum LDL increase

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~- RNA polymerase Ill transcribe transfer RNAs

and 5S ribosomal RNA
---
Three types of RNA polymerase in eukaryotic nuclei

Tn,e Location RNA synthe~1.cd Effect ofo-ananitin

I Nucleolus Pre-rRN A for 18, S.8 and 28S rRN As Insensitive

n Nucleoplasm Pre-mRN A, some snRN As Sensitive to 1 µg/ml
m Nucleoplasm Prc-tRN As, SS rRN A, some soRN As Sensitive to 10 µg/ml

Eukaryotic RN A

5. a-amanitin inhibits RNAP polymerase II & Ill

Three types of RNA polyn1erase in eukaryotic nuclei
Type Location RN A synthesiud Effect of ct-amanitin

I N ucleolus Prc-rRN A for 18, 5.8 and 28S rRN As Insensitive

11 N ucleoplasm Prc-mRN A, some snR.:.'JAs Sensitive to 1 µ.g/ml
m N uclcoplasm Pre-tRN As, SS rRN A, some snRN As Sensitive to 10 µg/ml

6 . UGA iITT Mitochondrial DNA is a codon tor Trvotoonan

~ I
Codon mtDNA translation code nDNA translation code

AUA Methionine lsoleucine

AGA Stop Arginine

AGG Stop Arginine

UGA Tryptophan Stop

AUG Methionine Methionine

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I . GENE KNOCK DOWN IS MEDIATED BY SIRNA

SiRNA's : silencening RNA , 21-25nt in length generated by selective nucleolytic
degradation of double stranded RNA's of ceelular or viral origin. SiRNAs anneal t o
various specific sites within target in RNAs leading to mRNA degradation , hence gene
knockdown

12. CRISPR/CAS9 is a genome editing tooj

' dsDNA
J

Cleavage

single gene can give rise to more than one protein by

ALTERNATIVE SPLICING
-c=/\- --.i_ --.a_ ___ CC-
=-.:::J_ _ _ _
Con1tlnnivr Splicing

Esoc1Sk.lppi•&

lntron Rrlrntlon

£1d nl,e£1ou

-c=I> I\
Allernalh·e 5' Splitt Silt

Allc:m11tlvr l ' Spike Sile

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. In the intestinal cells RNA editing helps in synthesis of

po B48 protein from apoBl00 mRNA, where a stop codon
enerated using the enz me CYTOSINE DEAMINASE.

6: o
orir:
cytosine
deamlnases
H, NJ' ..- H
___... ~ ,- ~I
0 N H
I I
Sugar Sugar
Cytosine (C) Uracil (U)

CAA changes to UAA - and it decreases to 48% - RNA editing

5. The component of RISC which degrade the target mRN~

is Arqonaute
a mlR N A - d•p•nd•n1: rac ru l «-m•nt

mR N A

1
neu C W I
m RN A

l
G e n o ~ll e ,, c l ng

Only methylated DNA is expressed

DAM - DNA methylase

o.Rypermefnylation of cm epG-1slands 1ITTne S'

regulatory portion of many genes DECREASE expression,
whereas hypomethylation IN CREASE it (increase or
e eas :?.

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7. CAP-Camp Activated protein c

itive regulator to induce transcription lac ope
Glucose absent, lactose absent:

cAMP Promoter
CAP )i-----------,-----,.---,---,
Operator /acZ lacY JacA
. I

RNA Polymerase~ Repre~or

No glucose in bacteria , increases cAMP + protein =CAP, if no repressor will move ahead

sex partners can't have a biologic

Hy related only_, to the m due to a ph 1

nomk lmpr,inting ,
{P) ~o

...~.,...
--
matmul
di wwww dlrCl!lffllftW
WW\'r l'J..Jc:i •\\ w m1)11111a4 PrUcT -\\'G !fflt

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1. Sanger sequencing is based on

chain-terminating dideoxy nucleotides by DNA polymerase
_ _ _ _ _d_u_ri_ng In vitro DNA re~G,,;l~ic;..;.;
a.;.;.
tio,;,.;n~ _ _ ___,

,
e
!NU muldplt coplti
e
attach a
e
add to four
0 ofasegmt nl primer polymtraH solutions
deNl1ure dsDNA

l1)1
ur.lnghul

e
grow complffllenbry
0
denalurate the
0
electrophorese the
chains undl iarmlnadon dyw grown chains four soludons
DJ J C G

.
-
G
G

- ...
- -
•
C
'
G

'
- - C
'
ddNTPs

2. A fusion of vector DNA ,bacterial promoters and SD

sequence with human cDNA (which allows eukaryotic
protein to be made in prokaryotic host cell ) is known as
Chimeric DNA
Human cell

·- ·--- EcoRI
Sticky
end
1-
>rti Recomblnt1n1
ONA
Gene for
human

u --
growth
Gene l o r h u man DNA hormone
growth hormone recombln,~1ion

Bocterlolc-ell .,._,.,
'-'-VTU -
Sticky_
o
nd
ilN A
Insertion

0
Bacteria I ~ , ._.,,,
chromosome 'lit'<1

Plasm Id
_9 BuctQrliJ I CQII for
contain ing gene for
O
human growth hormone

plasmid most important vector

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3. Mass spectromeffyis ttie tecnnique ofc lio1ce or

·nvestigation, screening and diagnosis of Inborn errors of
e b .

' t;
Sample • • • ••

-
_.
+ .
--
..
• • .
• •• · 1__,
• . ...
I

.... : I • • • ..
.
• •
e, • '•
.
•

Ionizer Mass Analyzer Detector

• MALDI • Time-Of- Flight (TOF) • Electron
• Electro-Spray • Quadrapole Multiplier
Ionization (ESI) • .Ion-Trap (EM)

. detecting molecular weight

~- CHIP is a widely used method for delineating the bindin
ite of a protein or small molecule on DNA or RNA.

~- p:~ , . . . .
~( Qnl"
N>4•_,•
J
...
Ni,k,,Qetf•ay
,_..,,.... _.
ONA ~ u a

( Gft--P•

Immunoprecipitating, protein DNA interaction

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s. Restriction fragment length polymorphism

is the main principle of DNA fingerprinting.
N ORMAL DllJ!AII! N ODW. DISEAH

-- --==
N ORMAL

-- -
Me4swtdi:'-a•

-- -- -
i l l
J

---- --==-
l>fl,ilr...-lt

I
---- ---- -
DIR.UE

. Mwtiian . . . . .
--1nr-•
.Ii l]
r- -j

..........
"'-v-'

Uses restriction endonucleases

6. A genomic liorary is a collection offragments

representing the entire genome of an organism, including
introns and regulatory sequences, whereas a cDNA library
is a collection of only the expressed sequences. (True
Vfalse ?l -TRUE r:::,~::::::,:-=-=::::~~============~
, 1.____ __,

r
84Ms Genomic Ubr.-y
.c:DN A Ubr4ry
A genomic: lib,.ry is I CQhcdion of A (ONA llbnry is I blend of cloned
the comple1t genomic DNA from one cDN.A fraom!f'ts 1nse1ted into a
Definition or911n1sm. ONA ts non!d In a sel!aion of host C211s, which
popuiattOft ol similar ,•eaou, eadl colledivety makes some plft of
con111mng ;, distinct in~ rt of DHA. the.- tr.inwiptamc ol the oro.inism.

b pre.-ioo Cnt1rc ~ e Only 1pcciftc genes.

I
Size ~er Smlller

h lUOflS Cu,r@nt Absent

It ulllazes plasmids, <XKtnid, l1mbd1 It 's no Intrans so uUltzH plasmids,

Vector ph~. YAC Md MC r« the pl\agt!l'lllds. lambda phage to
accommDdatlon ol l.trl)e fragmiertU . 11u:nmmod,ile niodi!S.t lrao111ent,..
- • - ---

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. ana ys1s 1s use to stu y po ymorp sms . n

is a genetic variant observable if the length of the
restriction fragment is altered by creating or abolishing a
restriction site or varying the number of short, tandem
otide r ats. (true_oL false)-JRU

--- ---
Normal Disuse Normal Disease
Normal

- -
t.htl IHtrktlon Sia.,

-- -- -
l l

• --- ---- -
D...st. w,Ar.ue
Olseue

--- -
--
...,...._n ••11~
on« 1nuk.1lon ,att

l
-
Cel electrophora,. Soulllem blot

8. RNA isolatea -from tissue is hybriaized to prooes for

specific genes that are fixed in a grid in small microscopic
spots on a chip. The above statement explain a technique
k/a Microarray and is used for comparative gene
expression analysis in 2 different tissues.

Name two other techn i ues of gene exRression anal sis?

1. Fluorescent in situ hybridization (FISH)

2. Reverse transcription polymerase chain reaction (RT-PCR)

. a restriction digest of the DNA from each individual is

eparated by gel electrophoresis, denatured, and
transferred (by blotting) to a membrane. A labeled probe is
used to detect the presence (or absence) of the target
equence. The technique of digesting, separating, blotting,
nd detecting DNA is known as Southern blotting.

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Mk:roat'fll)' DN A Soquo

0. Ch IP or chromatin
immunoprecipitation is
technique used
Protein-DNA
nteraction Ma
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MCQ
1. DNA methylation is not involved in
a) Capping
b) DNA replication
c) Gene inactivation
d) Mismatch repair
2. Double strand DNA breaks introduced by CRISPR/Cas9 are repaired by
a. Base Excision repair
b. Mismatch repair
c. N on homologous end joining repair
d. Homologous repair
3. The most common vector used in recombinant DNA technology is

a) Plasmid
b) cosmid
c) Phage
d) Bacterial artificial chromosome
4. In a double stranded DNA if Guanosine is 15%, how much is Adenine

a) 15%
b) 35%
c) 30%
d) 49%

5. Not true of Mitochondrial DNA I

a) double strand circular

b) code all the proteins ETC complexes
c) has heavy strand and a light strand
d) high rate of mutations

6. Silent mutations can be explained by the following property of genetic codon

a) Universal
b) degenerate

c) Non overlapping
d) commaless

7. RNAi mediates

a) Gene knockdown b) Gene knockup

d) Gene Knock in c) Gene knock out

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8. Telomerase is

1. RNA dependent DNA polymerase

2. DNA dependent DNA polymerase
3. RNA dependent RNA polymerase
4. DNA dependent RNA polymerase

9. Purines synthesis when compared to pyrimidine synthesis

a) Has no role of glycine

b) Has no role of folic acid
c) PRPP used in initial step
d) Is a mitochondrial pathway

10. The best technique to identify point mutations is

a) PCR
b) DNA sequencing
c) RFLP
d) Mass spectrometry

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