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ALL DATA FROM PAST PAPERS (2018-2022)

GROUP: BIOTECH BRAINY BUNCH

ADMINS: MEHWISH MUGHAL & BELLA

CONTACT: [email protected]

1. Write first step of RT-PCR? (2 marks)

First step of RT-PCR
 First strand reaction. Synthesis of CDNA using oligo dT primers (37°C) one hour.
 Second strand reaction - digestion of cDNA:RNA hybrid (RNaseH)-, Standard PCR with
DNA oligo primers.

2. Write down the principle of conventional PCR?(2)

A technique widely used in Molecular Biology and Biotechnology. Its name is from one of its
key component - DNA polymerase. As PCR progresses, DNA generated is itself used as
template for replication
3. What is use of Blank reaction and negative control in a PCR reaction? (2)
BLANK REACTION:
Controls for contamination contains all reagents except DNA template.
NEGATIVE CONTROL:
Controls for specificity of the amplification reaction contains all reagents and a DNA template
lacking the target sequence.
4. Write the extension rate and source of Taq Polymerase Vent and Pfu? (3 marks)
Polymerase Extension Rate (nt/'sec) Source Taq pol 75 T. T. aquaticus
 Vent >80 Thermococcus
 Vent >80 Thermococcus litoralis

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 Pfu 60 Pyrococcus furithen

5. Write down the principle of QRT-PCR? (3)

Quantitative real-time PCR is often confusingly known as RT-PCR (Real Time PCR) or RQ-
PCR. QRT PCR or RTQ-PCR are more appropriate contractions. QRT-PCR methods use
fluorescent dyes, such as Sybr Green, or Q-PCR is commonly used to determine whether a DNA
sequence is present in a sample and the number of its copies in the sample. fluorophore-
containing DNA probes, such as TaqMan, to measure the amount of amplified product in real
time.
6. Write note on Ligation mediated PCR? (3
METHODOLOGY:
Uses small DNA oligonucleotide linkers' (or adaptors) that are first ligated to fragments of the
target DNA. PCR primers that anneal to the linker sequences are then used to amplify the target
fragment
7. Write the chemicals used in conventional PCR and also reason of used of these
chemicals? (5)
MAJOR INGREDIENTS:
Microfuge tube, Thermal cycler, DNA template, Primers, Buffer, MgCI2, Distilled H20,
Deoxynucleotide triphosphates, DNA polymerase,

 MICROFUGE TUBES:
These are small cylindrical plastic tubes with conical bottoms.
 THERMOCYCLER:
The thermocycler works on the principle of Peltier effect, which raises and lowers the
temperature of the block in a pre- programmed manner. DNA POLYMERASES: Taq
polymerase, Pfu polymerase, Vent polymerase.

 TEMPLATE CAN BE DNA OR RNA:

Template DNA, RNA in case of reverse transcriptase. These include DNA cloning for
sequencing, gene cloning and manipulation, gene mutagenesis; construction of DNA-based
phylogenies, or functional analysis of genes; diagnosis and monitoring of hereditary diseases;
amplification of ancient DNA.
8. Differentiate the methodology and principles of Allele specific and AFLP PCR?
Allele-specific PCR used for identifying of SNPs. It requires prior knowledge of a DNA
sequence, including differences between alleles. Uses primers whose 3' ends encompass the
SNP. PCR amplification under stringent

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AMPLIFICATION WITH SNP SPECIFIC PRIMER:

Successful amplification with an SNP specific primer signals presence of the specific SNP in a
sequence. Conditions is much less efficient in the presence of a mismatch between template and
primer.
ALLELE SPECIFIC PCR:
This diagnostic or cloning technique is used to identify or utilize single-nucleotide
polymorphisms (SNPs).
AFLP PCR METHODOLOGY:
Genomic DNA is digested with one or more restriction enzymes. tetracutter (Msel) and a
hexacutter (EcoRI). Ligation of linkers to all restriction fragments. Pre-selective PCR is
performed using primers which match the linkers and restriction site specific sequences.
Electrophoretic separation and amplicons on a gel matrix, followed by visualization of the band
pattern. AFLP is a highly sensitive PCR-based method for detecting polymorphisms in DNA.
AFLP can be also used for genotyping individuals for a large number of loci.
9. Write the reagent components of PCR?
MAJOR INGREDIENTS:
Microfuge tube, Thermal cycler, DNA template, Primers, Buffer, MgCi2, Distilled H20,
Deoxynucleotide triphosphates, DNA polymerase,
10. How template is different in RT- PCR from conventional PCR?
RT-PCR is widely used in expression profiling to determine the expression of a gene. To identify
the sequence of an RNA transcript. A technique Widely used in Molecular Biology and
Biotechnology. Its name is from one of its key component - DNA polymerase. As PCR
progresses, DNA generated is itself used as template for replication.
11. Write the method of assembly PCR? (3)
Assembly PCR used to assemble two or more pieces of DNA into one piece. PRINCIPLE:
Assembly PCR is the synthesis of long DNA structures by performing PCR on a pool of long
oligonucleotides with short overlapping segments, to assemble two or more pieces of DNA into
one piece.
METHODOLOGY:
It involves an initial PCR with primers that have an overlap and a second PCR using the products
as the template that generates the final full-length product. ASSEMBLY PCR: Assembly PCR
used to assemble two or more pieces of DNA into one piece.
12. What are uses of Inter Sequence Specific PCR?
PCR used to produce a unique fingerprints of amplified product lengths.

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13. Write methodology of Ligation-mediated PCR?

Uses small DNA oligonucleotide 'linkers' (or adaptors) that are first ligated to fragments of the
target DNA. PCR primers that anneal to the linker sequences are then used to amplify the target
fragments.
14. What are uses of Ligation-mediated PCR?
DNA sequencing Genome walking DNA foot printing.
15. Where used Methylation Specific PCR?
MSP used in quantitative PCR provides information about methylation state of a given CpG
island.
16. Define colony PCR?
The screening of bacterial or yeast clones for correct ligation or plasmid products.
17. Write the methodology of the colony PCR?
The screening of bacterial or yeast clones for correct ligation or plasmid products. Pick a
bacterial colony with an autoclaved toothpick, swirl it into 25 ul of TE autoclaved dH20 in an
microfuge tube. a Heat the mix in a boiling water bath (90-1000C) for 2 minutes Spin sample for
2 minutes high speed in centrifuge. Transfer 20 ul of the supernatant into a new microfuge tube.
Take 1-2 ul of the supernatant as template in a 25ul PCR standard PCR reaction.
18. What are uses of real time PCR?
Real Time PCR is used to measure the quantity of a PCR product. The fluorescence, measured in
Real Time, is detected in a PCR cycler with an inbuilt filter fluorimeter. It is the method of
choice to quantitatively measure starting amounts of DNA, cDNA or RNA.
19. Define real time PCR?
Real Time PCR is a technique in which fluoroprobes bind to specific target regions of amplicons
to produce fluorescence during PCR.
20. What are advantage of nested PCR?
Very low probability of nonspecific amplification.
21. Define Multiplex PCR?
Multiplex PCR is a variant of PCR which enables simultaneous amplification of many targets of
interest in one reaction by using more than one pair of primers.
22. What are types of PCR?
Nested PCR, Multiplex PC, Multiplex PC, Touchdown PCR, Sequence-specific PCR, Reverse
transcriptase PCR, Long-range PCR, Whole-genome amplification, RAPD PCR (AP-PCR,
Quantitative real-time PCR.
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23. What is blank reaction?

Controls for contamination and contains all reagents except DNA o template.
24. What is negative control?
Controls for specificity of the amplification reaction contains all reagents and a DNA template
lacking the target sequence.
25. How many step of PCR reaction must write their names?
 Denaturation
 Annealing
 Extension

26. Define PCR?

A technique widely used in Molecular Biology and Biotechnology. PCR can be performed to
amplify DNA. It can be extensively modified to perform a wide array of genetic manipulations.
27. Mini primer per application polymerase name methodology?
Mini Primer PCR uses a thermostable polymerase (S-Tbr) that can extend from short primers as
short as 9 or 10 nucleotides.
28. ALU PCR? 2
The use of primers from a commonly repeated segment is called ALU-PCR, and can help to
amplify sequences adjacent (or between) these repeats.
29. WGA PCR or technique? 3
Whole Genome Amplification (WGA) is a method for robust amplification of an entre genome,
starting with nanogram quantities of DNA and resulting in microgram quantities of amplified
products. WGA has become an invaluable method for preserving limited samples of precious
stock material, particularly when using WGA methods that have been developed to amplify
material from a single cell.
30. Use of methylation in PCR?
Methylation-specific PCR is used to identify patterns of DNA methylation at CpG islands in
genomic DNA. Target DNA is first treated with sodium bisulfite, which converts unmethylated
cytosine bases to uracil, which is complementary to adenosine in PCR primers. Two
amplifications are then carried out on the bisulfite-treated DNA: One primer set anneals to DNA
with cytosines (corresponding to methylated cytosine), and the other set anneals to DNA with
uracil (corresponding to unmethylated cytosine). MSP used in quantitative PCR provides
information about methylation state of a given CpG island.
31. Describe denaturation step of PCR? (3)

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 First regular cycling event and consists of heating the reaction to 93°C - 98°C for 20- 45
seconds.
 It causes melting of DNA template yielding single strands of DNA.
Denaturation temperature
 Reduces double stranded molecules to single stranded.
 90-960C, 20-45 seconds.

32. ALU-PCR? (3)

The use of primers from a commonly repeated segment is called ALU-PCR, and can help to
amplify sequences adjacent (or between) these repeats.
33. How template is different in RT-PCR from conventional PCR?
In conventional PCR the template is DNA while in RT-PCR the template we use is RNA.
34. Methodology of QRT-PCR?
QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA
probes, such as TaqMan, to measure the amount of amplified product in real time.
35. Write first step of RT-PCR?
Assembly PCR is the synthesis of long DNA structures by performing PCR on a pool of long
oligonucleotides with short overlapping segments, to assemble two or more pieces of DNA into
one piece.
36. Write methodology of assembly PCR?
It involves an initial PCR with primers that have an overlap and a second PCR using the products
as the template that generates the final full-length product.
37. Difference between in situ PCR colony PCR and asymmetric PCR? (3)
 Colony PCR
The screening of bacterial or yeast clones for correct ligation or plasmid products.
 Asymmetric PCR
Asymmetric PCR is a variation of PCR used to preferentially amplify one strand of the original
DNA more than the other.
38. Principle of qRT PCR? (2)
The principle of qRT-PCR assays is straightforward following the RT of RNA into cDNA, it
requires a suitable detection chemistry to report the presence of PCR pro- ducts, an instrument to
monitor the amplification in real- time and appropriate software for quantitative analysis.

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39. Name 4 DNA modified enzymes and also explain it's uses. / Describe RM-System of
enzyme? (5)
At least four R-M systems are known
Type I
 Type I systems were the first to be characterized from E. Coli K12.
 The active enzyme consists of two restriction subunit, two modification subunit and one
recognition subunit.
 Type I systems are of little value for gene manipulation.
Type II
 Most of the useful R-M system is Type II.
 Type I enzymes recognize defined sequence and cut within it.
Type III
 Type Il enzymes have symmetrical recognition sequences but otherwise resemble type I
systems and are of little value.
Type lls
 Type Is systems have similar cofactors and structure to type Il but restriction occurs at a
distance from recognition site that limits their usefulness.

40. Application of PCR in medical science? (3)

 PCR is also used in molecular diagnostics and biochemical analyses. Among other things,
these techniques can be used in drug development, especially in measuring the efficacy of
drug therapy and research into cancer detection and treatment.
 PCR (polymerase chain reaction) tests are a fast, highly accurate way to diagnose certain
infectious diseases and genetic changes. The tests work by finding the DNA or RNA of a
pathogen (disease-causing organism) or abnormal cells in a sample.

41. What is Asymmetric PCR? (2)

Asymmetric PCR is a variation of PCR used to preferentially amplify one strand of the original
DNA more than the other.
42. Reverse Transcription PCR?
RT-PCR, also known as Reverse Transcriptase PCR, is a variation of the polymerase chain
reaction that typically measures RNA expression levels and also reverse transcribes the RNA
into DNA.
43. RAPD stands for?
Randomly amplified polymorphic DNA.
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44. What is RAPD Technique?

Randomly amplified polymorphic DNA (RAPD) is a PCR-based technique which uses arbitrary
primers which bind to the nonspecific sites on the DNA and amplify the DNA. These amplified
fragments are then migrated on agarose gel and difference in the band pattern is observed.
45. What is heat stable DNA polymerase?
A thermostable DNA polymerase is used in repeated cycles of primer annealing, DNA synthesis
and dissociation of duplex DNA to serve as new templates. During PCR, a heat stable DNA
polymerase, such as Taq polymerase an enzyme derived from the bacterium Thermus aquaticus.
46. Write down the steps of PCR?
PCR is based on three simple steps required for any DNA synthesis reaction:
 Denaturation of the template into single strands.
 Annealing of primers to each original strand for new strand synthesis.
 Extension of the new DNA strands from the primers.

47. What is Thermocycler?

The thermal cycler is a laboratory apparatus most commonly used to amplify segments of DNA
by the PCR. Thermal cyclers may also be used in laboratories to facilitate other temperature-
sensitive reactions, including restriction enzyme digestion or rapid diagnostics.
Micro centrifuge tubes or microfuge tubes are small, cylindrical plastic containers with conical
bottoms, typically with an integral snap cap. They are used in molecular biology and
biochemistry to store and centrifuge small amounts of liquid.
48. Define enzyme modification?
An enzyme that introduces minor bases into DNA or RNA or that alters bases already
incorporated.
49. Difference between linker and adaptor? (2)
The main difference between inker and adaptor is that former having blunt ends while later have
one cohesive end.
50. Difference between type-II and type-IIs?
Type II
 Most of the useful R-M system is Type II.
 Type I enzymes recognize defined sequence and cut within it.
Type lls

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 Type Is systems have similar cofactors and structure to type Il but restriction occurs at a
distance from recognition site that limits their usefulness.

51. Limitations of PCR?

 Contamination risk
 Primer complexities
 Primer-binding site
 Complexities
 Amplifies rare species
 Detection methods

52. Denaturation step?

 First regular cycling event and consists of heating the reaction to 93°C 98°C for 20-45
seconds.
 It causes melting of DNA template yielding single strands of DNA.

53. Differentiate between Nested and Multiplex PCR? Also write the applications of
Asymmetric PCR?
Nested PCR :
Nested PCR usually involves two sequential amplification reactions, each of which uses a
different pair of primers.
Multiplex PCR :
Multiplex PCR is a variant of PCR which enables simultaneous amplification of many targets of
interest in one reaction by using more than one pair of primers.
Application of Asymmetric PCR :
Asymmetric PCR can be used to form single stranded DNA from double stranded DNA, which is
then used for DNA sequencing in the mutagenesis method. Single stranded DNA is also
important for aptamer generation.
54. What is Hot Start PCR? (5)
This is a technique that reduces non- specific amplification during the initial set up stages of the
PCR. The technique may be performed manually by heating the reaction components upto the
melting temperature (e.g, 95°C). Before adding the polymerase, specialized enzyme systems
have been developed that inhibit the polymerase's activity at ambient temperature. This is done
either by the binding of an antibody or by the presence of covalently bound inhibitors that only
dissociate after a high-temperature activation step. DNA Polymerase- Eubacterial type DNA
polymerase, Pfu. These thermophilic DNA polymerases show a very small polymerase activity at
room temperature.

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55. Write down the formula for calculating melting temperature of primers?
Tm = 4(G+C) + 2(A+T)
56. Write the features of primers?
 Types of primers-random or specific
 Primer length
 Annealing temperature
 Specificity
 Nucleotide composition

57. Which type of primer is used for PCR?

PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers
designed specifically for the DNA region of interest.
58. Write the names of DNA polymerase which are mostly used?
 Taq Polymerase
 Pfu polymerase
 Vent polymerase
Taq: Thermus aquaticus (most commonly used)
Tfl: T. flavus
Tth: T. thermophilus
Tli: Thermococcus litoralis
Pfu: Pyrococcus furiosus (fidelity)
59. What is conclusion PCR?
PCR can be performed to amplify DNA. It can be extensively modified to perform a wide array
of genetic manipulations.
60. Write the names of any 5 types of PCR?
 Nested PCR
 Multiplex PCR
 Real-time PCR
 Quantitative PCR
 Arbitrary Primed PCR
 High-fidelity PCR
 Fast PCR
 Hot-start PCR
 GC-rich PCR
 Long-range PCR

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61. Primer requirements? (3)

 Length of 18-24 bases.
 40-60% G/C content.
 Start and end with 1-2 G/C pairs.
 Melting temperature (Tm) of 50-60°C.
 Primer pairs should have a Tm within 5°C of each other.
 Primer pairs should not have complementary regions.

62. Principle of asymmetric PCR? (3)

The use of an excessive amount of single-strand (target single-strand) specific primer amplifies
only a single template ssDNA for DNA sequencing and hybridization probing. It uses a 1:10
volume of primers in which a 10 times higher volume of target-specific primer is used.
63. Define primer dimers? 2
A primer dimer is a potential by-product in the PCR. It is a common biotechnological method. A
primer dimer consists of two primer molecules that have attached to each other because of
strings of complementary bases in the primers.
64. Application of suicide PCR? 2Marks
Suicide PCR is typically used in paleogenetics or other studies where avoiding false positives
and ensuring the specificity of the amplified fragment is the highest priority.
65. Function of DNA ligase enzyme? 3Marks
DNA ligases play an essential role in maintaining genomic integrity by joining breaks in the
phosphodiester backbone of DNA that occur during replication and recombination, and as a
consequence of DNA damage and its repair.
66. Applications of In Situ PCR? 3Marks
PCR is an extremely sensitive technique which amplifies single copy gene sequence to high
levels, easily detectable by ISH. Thus, this new technique could have important applications in
infectious disease and oncology. Studies on in situ PCR detection of viral DNA sequences
include HIV, HPV, CMV, and HBV.
67. What is Assembly PCR? (2)
Assembly PCR used to assemble two or more pieces of DNA into one piece.
68. Western blotting procedure?
A technique used to detect the presence of a specific protein in a complex protein mixture.
 Sample preparation
 Gel Electrophoresis
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 Blotting (or transfer)

 Blocking
 Antibody probing
 Detection

1. The choice of extraction method depends primarily on the sample and whether the
analysis is targeting all the proteins in a cell or only a component from a particular
subcellular Traction.
2. Samples are loaded into separate wells. A protein marker is also loaded. The separated
protein mixtures are transferred to a solid support for further analysis.
3. Transfer can be done in wet or semi-dry conditions. Semi-dry transfer is generally faster.
Wet transfer is recommended for large proteins, >100 kD.
4. Blocking is a very important step in the immunodetection phase of Western blotting
because it prevents non-specific binding of antibody to the blotting membrane.
Protein of interest is detected and localized using a specific antibody. Western blotting protocols
utilize a non-labelled primary antibody directed against the target protein. A species-specific,
labelled secondary antibody directed against the constant region of the primary antibody is then
used. The most common antibody label used in Western blots is HRP. The signal is detected
when HRP is exposed to a substrate solution in the final step of the immunodetection procedure.
69. Properties of DNA probe?
DNA probes are used for detecting nucleic acid sequences of interest. They are useful for the
identification of microorganisms, the diagnosis of infections, and the identification of antibiotic-
resistant genes in patients, among other applications.
70. Nomenclature of restrictions enzyme. Also explain mini primer PCR?
Nomenclature of restrictions enzyme :
 A suitable system was proposed by Smith and Nathans (1973).
 The species name of host organisms is identified by the first letter of genus and first two
letters of specific epithet. E. coli = Eco , H. influenzae = Hin.
 Strain identification is written as EcoK.
 In case, host strain has several restriction and modification systems, these are identified
by roman numerals for example, in case of H. Influenzae Hindl, Hindll, Hindlll etc.
 All restriction enzymes have general name endonuclease R and modification-methylase
M followed by the system name, for example, in case of H. Influenzae R. Hindlll or M.
Hindlll.
Mini primer PCR :
Mini Primer PCR uses a thermostable polymerase (S-Tbr) that can extend from short primers as
short as 9 or 10 nucleotides. This method permits PCR targeting to smaller primer binding

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regions, and is used to amplify conserved DNA sequences, such as the 16S or eukaryotic 18S)
rRNA gene. PCR that can extend from short primers.
71. Calculate restriction sites every four, six, eight?
Calculate the amount of each that you need to add to a restriction digestion in order digest 5ug
(5000ng) of DNA with 5 units of enzyme. For example if my DNA is at 190 ng/ul, I would need:
5000ng/190ng/ul = 26 ul of my sample.
72. What is Nested PCR?
Nested PCR usually involves two sequential amplification reactions, each of which uses a
different pair of primers.
73. What is the In situ PCR? (2)
In Situ PCR (ISH ) is a polymerase chain reaction that actually takes place inside the cell on a
slide. In situ PCR amplification can be performed on fixed tissue or cells. Applies the
methodology of hybridization of the nucleic acids. Allows identification of cellular markers.
Limited to detection of non-genomic material such as RNA.
74. Methylation PCR method?
Methylation-specific PCR is used to identify patterns of DNA methylation at CpG islands in
genomic DNA. Target DNA is first treated with sodium bisulfite, which converts unmethylated
cytosine bases to uracil, which is complementary to adenosine in PCR primers. Two
amplifications are then carried out on the bisulfite-treated DNA: One primer set anneals to DNA
with cytosines (corresponding to methylated cytosine), and the other set anneals to DNA with
uracil (corresponding to unmethylated cytosine).
75. Mechanism of DNA ligase?
DNA ligase is a specific type of enzyme, a ligase, that facilitates the joining of DNA strands
together by catalyzing the formation of a phosphodiester bond. It plays a role in repairing single-
strand breaks in duplex DNA in living organisms, but some forms may specifically repair
double-strand breaks. The mechanism of DNA ligase is to form two covalent phosphodiester
bonds between 3' hydroxyl ends of one nucleotide ("acceptor"), with the 5' phosphate end of
another ("donor"). Two ATP molecules are consumed for each phosphodiester bond formed.
76. Amplification with SNP specific primer?
Successful amplification with an SNP-specific primer signals presence of the specific SNP in a
sequence.

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