Supplementary Information to Chapter 49

MOLECULAR BIOLOGY APPLICATIONS AND TECHNIQUES

Applications of Molecular Biology in Clinical Medicine
1. Diagnosis of molecular diseases and infections
2. Application of recombinant DNA technology in production of proteins for replacement therapy
3. Understanding the inheritance and pathogenesis of genetic disorders
4. DNA fingerprinting in medico legal practice
5. Development of gene therapy.

Production of Proteins for Replacement Therapy

¾¾ Recombinant growth hormone is used to treat children with growth hormone deficiency (GHD)
¾¾ Recombinant leptin has been used in treatment of childhood obesity
¾¾ Epoetin is widely used in treatment of chronic kidney disease (CKD)
¾¾ Simple replacement of adrenocorticotropic hormone (ACTH) is not possible since it is produced as a precursor which
is cleaved to give active ACTH.
¾¾ Replacement with target hormone is more useful and effective.

What is cDNA?
¾¾ Complementary DNA is prepared from messenger RNA using Reverse transcriptase
¾¾ Complementary DNA has only coding sequences
¾¾ Genomic DNA has both coding and noncoding sequences
¾¾ Presence of messenger RNA indicates the presence of viable (living) organisms
¾¾ Complementary DNA can also be used to prepare gene libraries and recombinant DNA

NAT for HIV infection

¾¾ Nucleic acid testing (NAT) is now used in the early detection of HIV infection
¾¾ Reduces the window period
¾¾ Presence of infection can be detected as early as 2 to 3 weeks
¾¾ HIV-DNA can be isolated from leukocytes during seronegative stage from infected individuals
¾¾ Helps early treatment, donor detection and monitoring progress
¾¾ Also helps to detect genes producing drug resistance.
 Supplementary Information to Chapter 49 171

What is restriction mapping?

¾¾ Different restriction endonucleases cleave DNA at different but specific sequences
¾¾ Cleaving the same stretch of DNA with different enzymes will give restriction fragments of differing length
¾¾ Using a set of overlapping fragments, a restriction map of the particular stretch of DNA can be produced.
¾¾ The presence of alterations in sequence (even SNP) can be detected by RFLP
¾¾ Identifying a specific sequence on DNA
¾¾ A labelled DNA probe is used to identify the sequence.

Southern blotting
¾¾ Initially the DNA is subjected to RE (restriction endonuclease) cleavage
¾¾ The fragments are separated by agarose gel electrophoresis
¾¾ Gel is blotted so that the nitrocellulose membrane has the same imprint
¾¾ Labelled probe is added
¾¾ Hybridization occurs by complementary base pairing
¾¾ Detected depending on nature of the probe-fluorescent probes easiest to detect
¾¾ Originally radioactive probes were used.

Applications of Southern Blotting

¾¾ Detect specific mutations
¾¾ DNA fingerprinting uses the southern blot analysis of two samples for comparison
¾¾ Single nucleotide polymorphism can also be detected
¾¾ Evolutionary studies: purified mitochondrial DNA has been subjected to restriction mapping
¾¾ Degree of divergence can be calculated from the rate of base substitutions which is 10 times more in mtDNA than in
nuclear genome. Divergence indicates origin from a common ancestor
¾¾ Evolutionary relationships between species, migration pattern of people and different types of mutations can be
studied.

Uses of Restriction Endonucleases

1. To prepare rDNA molecules
2. To prepare DNA fragments for Southern blotting
3. RFLP
4. PCR using rDNA.

Polymerase Chain Reaction (PCR)

¾¾ In vitro amplification of a specific DNA sequence which revolutionized diagnostic molecular biology
¾¾ Kary Mullis (Nobel prize winner) developed the technique
¾¾ Process carried out at high temperature
¾¾ Uses a thermostable enzyme, Taq Polymerase from thermus aquaticus
¾¾ DNA primers of flanking sequence of DNA sequence to be amplified are required.

Steps of PCR Technique

¾¾ PCR mixture contains target sequence to be amplified, dNTPs, Taq polymerase and primers
¾¾ Denaturation of DNA at 950C
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¾¾ Annealing of primers at 550C

¾¾ Amplification at 720C
¾¾ Detection of the specific sequence by hybridization or sequencing
¾¾ For diagnostic purposes, detection by hybridization is sufficient, but for genetic studies sequencing is required.

Quantitative PCR or Real time PCR

¾¾ It is an automated process where the number of gene copies after each cycle can be calculated
¾¾ Very commonly used for infectious diseases (Hepatitis, HIV) to assess the severity of infection and response to
treatment
¾¾ Useful in detecting drug resistance also.

Reverse Transcriptase (RT)

¾¾ RNA dependent DNA polymerase present in retroviruses
¾¾ Temin and Baltimore identified the enzyme RT in tumor viruses
¾¾ Makes a DNA copy of the viral RNA to form a RNA-DNA hybrid
¾¾ RNA is hydrolysed by RNase H
¾¾ Complementary copy of the DNA strand is synthesised
¾¾ The new DNA strand is integrated to host cell DNA.

Uses of RT
1. In RT-PCR
2. Use of retroviral vectors for gene therapy
3. To prepare cDNA.

Nested PCR
¾¾ Modified PCR technique when DNA to be amplified is present in low concentrations.
¾¾ Spurious sequences may also be amplified
¾¾ To increase the specificity of amplification, nested PCR is done
¾¾ 10-20 cycles of PCR done by one set of primers
¾¾ A second PCR is done with a different set of primers which are nested between the first set of primers
¾¾ Clinical application is to detect microchimerism
¾¾ To detect donor derived white blood cells in recipient’s blood by detecting genetic polymorphism of HLA–DR region
of MHC complex.

Multiplex PCR
¾¾ A technique used to directly amplify a gene of interest without cloning and subcloning
¾¾ Multiple pairs of oligonucleotide primers are used
¾¾ Direct sequencing of the multiplex PCR products after electrophoretic separation detects point mutations in patient’s
gene
¾¾ Used in identifying different mutations, e.g. in Lesch–Nyhan syndrome
¾¾ Different types of mutations have been detected in the HGPRTase gene.
¾¾ Used in chromosome walking technique (described below)
¾¾ Identification of the major mutation helps in prenatal and clinical diagnosis
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¾¾ The CFTR gene has been cloned into an expression vector along with its control elements and it was fully expressed
in mouse culture cells.

Telomerase-The mitotic clock

¾¾ When DNA replicates, the ends are not replicated
¾¾ These ends called telomeres have repetitive sequences
¾¾ After each cell division the telomeres have a tendency to get shortened
¾¾ The enzyme telomerase prevents shortening of telomeres
¾¾ Adult cells have low telomerase activity so that a cell automatically stops dividing after a certain number of divisions
¾¾ Cancer cells have persistent or reactivated telomerase activity that makes them immortal.

DNA Sequencing
¾¾ Analysis of the sequence of nucleotides in DNA
¾¾ Sufficient DNA for sequencing can be produced by PCR
¾¾ Sanger’s procedure is based on interrupted enzymatic cleavage (See below)
¾¾ Sequencing can also be done by chemical cleavage approach by Maxam and Gilbert
¾¾ Dye terminator sequencing
¾¾ Helps to detect SNPs –HapMap of millions of SNPs already generated. HapMap is a database that describes common
patterns of genetic variation affecting health, disease, response to drugs and environment
¾¾ May help direct correlation of mutations with clinical status of patients.

Steps of DNA Sequencing

¾¾ DNA region of interest is inserted into bacteriophage
¾¾ The rDNA Molecule thus produced is purified
¾¾ The known sequence of the bacteriophage downstream of the DNA insert serves as a hybridization site for a universal
primer
¾¾ Extension of this primer using DNAP, all for dNTPs and one ddNTP helps to interrupt the elongation when ddNTP is
added. Dideoxy analog (ddNTP) of the different nucleotides are used. When these are incorporated into the growing
DNA chain, further elongation is prevented. The medium contains both dNTP and ddNTP, so that the growing DNA
molecule will be terminated at random points
¾¾ The fragments of end labelled DNA are now separated by electrophoresis
¾¾ The DNA sequence of each fragment is then determined.

Gene Libraries
¾¾ Genes are separated by RE cleavage and inserted into expression vectors
¾¾ cDNA can be prepared and used for cloning
¾¾ These rDNA molecules are cloned, the bacterial clones are selected by insertional inactivation of bacterial resistance
genes
¾¾ Different rDNA molecules present in the vectors form the gene library.

Molecular Cloning
¾¾ Cleavage of vector and DNA insert using same RE
¾¾ Annealing and Ligation of ends by DNA ligase
¾¾ Insertion of rDNA into bacteria
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¾¾ Some bacteria take up the recombinant plasmid

¾¾ Others may take up the plasmid without DNA insert
¾¾ The antibiotic resistance genes in plasmid are inactivated by RE cleavage
¾¾ Bacterial colonies can be selected by insertional inactivation and by replica plating
¾¾ The selected colonies are further subcultured to produce human proteins in large amounts.

Gene Transfer
¾¾ Normal genes may be transferred using cloned rDNA molecules into cells with defective genes
¾¾ Different vectors can be used
¾¾ cDNA introduced into cloning vectors have been successfully used
¾¾ Other vectors are modified retroviruses, adenoviruses and liposomes.

Nick Translation
¾¾ Nick translation produces labeled probes
¾¾ Single strand breaks, nicks are produced by the random cleavage of a phosphodiester bond by DNAse I
¾¾ Radioactive (32P labeled) dNTPs are incorporated into the DNA strand by DNAP
¾¾ The DNA is derived from cloned DNA, cDNA or viral DNA.

Random Primer labeling

¾¾ Requires only very small quantities of DNA
¾¾ Can label longer DNA strands and produce labeled probes
¾¾ The labeled probe is hybridized with random hexanucleotides to serve as primers for DNA synthesis
¾¾ The enzyme used is Klenow enzyme.

Blue White Screen

¾¾ The vector has Lac Z gene
¾¾ Insertion of DNA of interest disrupts the gene
¾¾ If the DNA has not been taken up by the vector, the gene is intact, the gene product is transcribed and translated
¾¾ As a result the colonies produced will be blue in color
¾¾ The colonies produced by vector having DNA insert will be white in color.

In Situ Hybridization
¾¾ A simple technique to detect expression of a specific gene
¾¾ Tissue of interest is fixed, mounted on to microscopic slide
¾¾ Proteins are digested by suitable proteases
¾¾ Labeled probe added to hybridize with cellular mRNA (flourescent label)
¾¾ Helps to identify genes which are expressed.

Nuclease Protection Assay (NPA)

¾¾ Suitable for simultaneous analysis of mRNA species
¾¾ RNA sample in solution is hybridized with labeled probes
¾¾ Any remaining unhybridized probe is hydrolyzed by nuclease
¾¾ The hybridized probe is then separated through gel electrophoresis
¾¾ NPA is commonly used to detect RNA transcripts in different tissues.
 Supplementary Information to Chapter 49 175

Nonsense mediated decay (NMD)

¾¾ Cytoplasmic mRNAs are broken down in the P bodies by cellular RNAses.
¾¾ Initial deadenylation of the poly A tail and decapping of the 5’ cp makes the RNA susceptible to the action of
exonucleases.
¾¾ RNAs which have a nonsense mutation which cannot produce a functional protein are degraded without translation
by NMD.
¾¾ This is a surveillance mechanism to prevent translation of mistranscribed or mis processed mRNAs.

Single Strand Conformation Polymorphism (SSCP)

¾¾ Single nucleotide polymorphism at restriction sites can produce RFLP
¾¾ SNP occuring at other sites can be detected by SSCP
¾¾ Single strand DNA fragments less than 400 nucleotides in length migrates on electrophoresis depending on its
conformation
¾¾ DNA or cDNA can be amplified by PCR and then SSCP
¾¾ Genes associated with Long QT syndrome and SIDS were analyzed by this technique
¾¾ Based on previous knowledge of the gene sequence.

Steps of Chromosome Walking

¾¾ Defines the arrangement of genes along long stretches of DNA
¾¾ Several techniques are combined
¾¾ A phage library is screened with a DNA or RNA probe to select a sequence of interest
¾¾ Selected probe is cloned, restriction mapped and a small fragment subcloned and labelled by nick translation
¾¾ This labeled probe is used to rescreen the phage library for complementary sequences and these are cloned
¾¾ The process is repeated till the gene of interest is reached
¾¾ Used to identify the CF gene located on the long arm of Chromosome 7 about 250kb long.

Site Directed Mutagenesis

¾¾ Controlled alteration of a selected region of DNA
¾¾ May involve substitution, insertion or deletion of specific nucleotide sequences
¾¾ Cloning of the altered gene is done
¾¾ Helps to identify the role of specific DNA sequences-regulatory role
¾¾ Used in detecting the role of a particular type of mutation in the phenotypic expression of a disease
¾¾ Example, dystrophic epidermolysis bullosa due to mutations in type IV collagen
¾¾ Mutation corresponding to abnormal phenotype can be detected.

Retrovirus Vector used in Gene Therapy

¾¾ Retroviruses which cannot replicate within the host cell
¾¾ Can carry only small genes
¾¾ May disrupt the host cell genome during integration
¾¾ Illegitimate combination can occur
¾¾ But once inserted it is permanent since the gene integrates with host cell chromosome.
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Other Vectors used in Gene Therapy

¾¾ Adenoviruses have more affinity for the cells they infect
¾¾ Can carry longer genes
¾¾ Effect is only transient
¾¾ Liposomes can be used to target the gene to a specific tissue since they can pass through cell membranes.

DNA Profiling (DNA Fingerprinting)

¾¾ DNA fingerprinting introduced in 1984 by Sir Alec Jeffreys
¾¾ A reference sample collected by buccal swab and compared with suspected sample
¾¾ Samples of biological relatives can provide an idea
¾¾ Uses highly repetitive sequences-variable number tandem repeats (VNTR) called short tandem repeats (STRs)
¾¾ VNTR loci similar in blood relatives but very different in unrelated persons
¾¾ Originally RFLP analysis and comparison of Southern blot was done
¾¾ Now PCR amplification of specific STRs done
¾¾ Mutiple STR loci compared, at least 13 loci
¾¾ Number of repeats different in different persons
¾¾ Quicker results available.

FOLLOWING ARE A FEW MCQS, BASED ON MOLECULAR BIOLOGY TOPICS

1.  Which of the following is not true regarding polymerase chain reaction?
A. An in vitro technique of DNA amplification
B. Can amplify either genomic DNA or cDNA
C. Useful in screening for HIV
D. Simplest technique of DNA sequencing.
Ans. 1-D

2.  Restriction mapping is used to detect

A. Bacterial infections
B. Regulatory elements within DNA
C. Genetic polymorphism
D. Number of gene copies in a patient.
Ans. 2-C

3.  Which of the enzymes listed is present in human beings?

A. Restriction endonucleases
B. Telomerase
C. Taq polymerase
D. Reverse transcriptase.
Ans. 3-B
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4. Sanger’s DNA sequencing technique uses

A. Restriction endonuclease cleavage followed by PCR
B. PCR and interrupted enzymatic cleavage
C. Nested PCR
D. Quantitative RT-PCR.
Ans. 4-B

5. Which of the following processes is non enzymatic?

A. Cleavage of DNA at specific sites
B. Annealing of primers
C. Joining the vector to the DNA insert
D. Adding polyA tail to blunt ends.
Ans. 5-B

6.  Recombinant DNA molecules cannot be used for

A. Preparation of gene libraries
B. Molecular cloning to produce proteins
C. Gene transfer for therapeutic purposes
D. Preparation of monoclonal antibodies.
Ans. 6-D

7.  Which of the statements regarding plasmids is not correct?

A. Associated with large chromosomes
B. Possess antibiotic resistance genes
C. Replicate independently of bacterial chromosome
D. Several copies of the plasmid are present in a bacterial cell.
Ans. 7-A

8.  The technique used to identify bacterial colonies with recombinant plasmids is
A. Nick translation
B. Random primer labeling
C. Blue white screen
D. In situ hybridization.
Ans. 8-C

9.  The process which identifies SNP is

A. Nuclease protected assay
B. Nonsense mediated decay
C. Single strand conformational polymorphism
D. Reverse transcriptase PCR.
Ans. 9-C
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10.  Chromosome walking involves all the techniques listed EXCEPT

A. Labeling of a DNA fragment
B. Restriction mapping
C. Cloning of target DNA
D. RNA interference.
Ans. 10-D

11. Given below are pairs of disease and techniques used to study the molecular defect. Pick
out the mismatched pair
A. Site directed mutagenesis and Huntington’s disease
B. DNA sequencing and Bloom syndrome
C. Multiplex PCR and Lesch Nyhan syndrome
D. Chromosome walking and Cystic fibrosis.
Ans. 11-A

12.  Which of the hormones listed is not produced by rDNA for replacement?
A. Growth hormone
B. Leptin
C. Erythropoietin
D. Adrenocorticotropic Hormone.
Ans. 12-D

13.  The protein which is not a transcription factor is

A. MAPK
B. SMAD
C. STAT
D. NFkB
Ans 13-A

Explanation
MAPK (Mitogen Activated Protein Kinase)
These are serine/threonine kinases similar to CDKs. They are activated by a phosphorylation cascade mediated by
a variety of stimuli like proinflammatory cytokines, stress, heat shock, mitogen, etc. They regulate gene expression,
proliferation, differentiation, mitosis and apoptosis by activating other proteins by their kinase activity.

JAK-STAT (Signal Transducers and Activators of Transcription)

STATs remain inactive in cytoplasm
Phosphorylation of SH2 domains by JAKs
Undergo dimerization
Dissociate from receptor
Translocate to nucleus
STATS activate specific gene regulatory elements leading to transcription of genes.
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SMAD-Transforming Growth Factor Beta

Unique type of cytokine receptor
Possesses intrinsic serine/threonine kinase activity
Enzyme activity in cytoplasmic signaling domain
SMADs are transcription factors that regulate expression of genes
Activated by phosphorylation.

NFkB
Activation of NFkB occurs by signal induced degradation of IkB protein
Phosphorylation of IkB protein regulatory domain by IkB kinase (IKK)
IkB inhibitor molecules are ubiqutinated and the protein is degraded.
NFkB is now free to enter the nucleus and turns on transcription of proteins responsible for innate and adaptive immune
response
An auto feed back loop operates by expression of a repressor of NFkB.

14.  The genetic modification having a negative therapeutic effect is

A. Exon skipping
B. Reversal of hemoglobin switch
C. Gene amplification
D. Gene silencing.
Ans. 14-C

Explanation
Exon Skipping
A technique used to restore a mutant reading frame by skipping an exon having a premature stop codon
As a result of this mutation, a truncated protein is produced which is non functional
Leads to DMD where dystrophin is defective or deficient
By skipping one exon by alternative splicing, the reading frame is restored
As a result a partially functional protein is produced
A less severe form of the disease, Becker’s muscular dystrophy instead of DMD is the result.

Gene Amplification
Gene amplification occurs in some cases following drug therapy
When methotrexate is given the enzyme folate reductase is competitively inhibited
Inhibition of folate reductase is the basis of therapeutic effect of the drug
On continued administration of the drug, the gene for folate reductase is amplified
This decreases the therapeutic effect.

Gene Silencing
Micro RNAs and small interfering RNAs are present in cells which regulate transcription and translation
This is done by both RNA silencing and RNA interference
Large hairpin RNAs are processed in the nucleus and cytoplasm by a DICER (dsRNA dicing action) complex to form
RNA induced silencing complex (RISC). DICER is an endoribonuclease
The RISC guides the miRNA to bind to complementary sequence on mRNA and represses translation
Chemically synthesised siRNA can mimic microRNA and is being tried in therapy.
180 Textbook of Biochemistry— Case Studies

15.  The response element present on RNA is

A. cAMP response element-binding (CREB)
B. Iron response elements (IRE)
C. Sterol regulatory element binding (SREB)
D. Thyroid response elements (TRE)
Ans. 15-B

Explanations
CREB-cAMP Regulated Proteins
Regulate the expression of genes that contain cAMP regulatory elements (CRE)
Proten Kinase A activated by cAMP phosphorylates CREB leading to activation
Hormone induced long term changes are thus brought about.

IRE-Iron Response Elements

Translational regulation of iron metabolism in humans is dependent on RNA secondary structure. Ferritin and transferring
receptor expression levels are reciprocally regulated in their response to changes in iron levels. When iron is scarce, the
amount of transferring receptor increases and no ferritin is synthesized. Ferritin mRNA has a stem loop structure at its
5’ untranslated region. This loop binds the IRE binding protein (IRP) that blocks translation as IRE binds on the 5’ side
of the coding region. When iron level increases, IRP bound to iron cannot bind RNA. Thus ferritin mRNA is translated
since it is released from IRP. The ferritin will sequestrate the excess iron.

SREBP-Sterol Regulatory Element Binding Protein

Sterol response elements on DNA are regulated by binding of Steroid hormone receptor complex which causes
transcription of specific genes
Proteins produced will bring about the metabolic effects.

Thyroid Response Elements (TRE)

The TRE is on DNA when bound to thyroid hormones will be trancsriptionally regulated.

16. Which of the processes listed leads to production of animals which have both alleles from
the same parent?
A. Gene knock out
B. Stem cell transplantation
C. Producing a transgenic animal
D. Somatic cloning.
Ans. 16-D

Explanations
Gene Knock out
A specific gene is “knocked out” or rendered nonfunctional in an intact organism
A null or knock out mouse is created by introducing a purified recombinant gene into modified embryonic stem cells so
that it replaces the normal gene.
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The ES cell thus modified is injected into a blastocyst

Gene knockout or transgenic animal
The blastocyst is implanted into a surrogate mother
The offspring with altered gene is selected
They can be bred so that only offspring homozygous for the altered knockout gene is produced
The technique helps to identify the exact role of the inactivated gene.

Somatic Cloning
The first cloned animal produced from adult mammalian cells was Dolly
Mammary gland cells from a Finn Dorset ewe (6 years old) were cultured.
These cells were arrested in G0 phase.
Nuclei from these quiescent cells transferred to enucleated oocyte from a Scottish blackface ewe
This artificially fertilized egg now developed into a morula or blastocyst stage
Surrogate mother. This blastocyst was then transferred to the womb of a surrogate mother ewe who carried the fetus to
term
Dolly was born and grew up, but was euthanasised in 2004 due to a progressive lung disease associated with premature
ageing. Ethical questions as to decision making about genetic make up and surrogacy were raised.
However it has opened the doors to therapeutic cloning to create embryonically tailored cells to suit the requirements
of particular patient. Being explored and developed for organ cloning and transplantation even from stored cord blood.

17.  Newborn screening is not beneficial in

A. Mitochondrial cytopathies
B. Phenylketonuria
C. Galactosemia
D. Congenital hypothyroidism.
Ans. 17-A.

Explanation
Mitochondrial Cytopathies
A 90% of mitochondrial proteins are nuclear encoded
Only 13 proteins are coded by mitochondrial DNA
Generally the ETC is defective which leads to lactic acidosis, deficient energy production with resultant muscular
weakness and exercise intolerance
Maternal inheritance is the hall mark
Clinical manifestations often set in at a later age, depending on the amount of mutant DNA in a given cell or tissue
The number of mitochondrial gene copies is high and exhibits heteroplasmy
Higher the content of mutant genes, earlier and more severe the symptoms
The clinical severity will differ in siblings with same disorder.

18.  Retroviral vectors have the advantage that

A. Large genes upto 36kb can be transferred
B. The new gene is integrated into the host cell chromosome
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C. Prevents disruption of host cell gene

D. The integration occurs only when the host cell undergoes division.
Ans. 18-B

19.  The technique used to analyze thousands of genes simultaneously is

A. DNA profiling
B. Therapeutic cloning
C. DNA microarray
D. Antibody microarray.
Ans. 19-C

Explanation
DNA Microarrays
Gene specific oligonucleotide probes are immobilized on a solid matrix (chip)
Thousands of unique probes are thus made available at a specific space
Labeled target nucleic acids derived from cells of an organism are treated with these chips
Hybridization is detected by the emission of fluorescence
The target can be immobilized at a specific site on the chip and even quantitated
High resolution imaging and computer analysis
Rapid screening of DNA for disease associated mutations can be done
The presence of sequence changes in ER positive and negative breast cancer patients was studied using microarray and
genetic changes in cDNA used for prediction of prognosis.

Antibody Microarray
Proteomic analysis by antigen antibody interactions on similar immobilized surfaces
The protein microarrays can capture target proteins using antibodies
Aptamers which are short stretches of RNA/DNA that can bind specific proteins are also being developed
The protein arrays are developed using recombinant proteins
Used in treatment of Glioblastoma multiformi
In GBM the Abnormal activation of PI3 Kinase pathway occurs by activation by RTKs.
Specific RTK inhibitors can be identified and used in therapy.

20.  Cisplatin acts as an antitumor agent by

A. Inhibiting nucleotide synthesis
B. Changing the shape of DNA
C. Arresting cell division
D. Inducing apoptosis.
Ans. 20-B

Explanation
Cisplatin forms cross links with DNA
Bent structures of the cisplatin–DNA adducts are recognized by several DNA binding proteins such as NER proteins.
A combination of cisplatin, bleomycin and vinblastin was found to be very effective in disseminated testicular cancer
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Later bleomycin is replaced by etoposide

Tour de France- Leading cyclist Lance Armstrong won the event 7 times after treatment

21.  Which of the markers listed is useful in prenatal screening?

A. CK-BB
B. AFP
C. LAP
D. IMA
Ans. 21-B

Explanation
Prenatal screening done twice; first and second trimester (15 and 22 weeks)
First trimester screening is done with PAPPA and beta hCG and NT.
Low AFP and unconjugated estriol and high beta hCG and dimeric inhibin A are seen in trisomy 21. Ultra sonography
is also helpful.

22.  All are common features of genetic disorders EXCEPT

A. Dominant negative effect
B. Genetic anticipation
C. Acquisition of a novel function
D. Haploinsufficiency.
Ans 22-C

Explanation
Acquisition of a novel function
Occurs when the mutation leads to cancer
p53 mutations have been found to result in oncogenic gain in function
Dominant negative effect due to mutant induced co-aggregation of p53 and its paralogs.

23.  The metabolic profile of inborn errors is studied by

A. DNA sequencing
B. Polyacrylamide electrophoresis
C. Tandem mass spectrometry
D. DNA microarrays.
Ans. 23-C

Explanation
Tandem mass spectrometry
Revolutionized the study of metabolic disorders
Quick and effective separation of metabolites which are structurally very similar has established the existance of several
in born errors
Diagnosis of organic acidurias, aminoacidopathies, defective proteins and peptides are all possible.