MICR5831 L5: PCR and Sequencing Data 7/29/25

Describe the features of primers (slide 3)

-Short sequences of ssDNA that match the template DNA

-Generally 20-30 nucleotides long

-Bind complementary sequences in a single-stranded template

What are the components in a PCR reaction (slide 3 and 4)

1) Template DNA (any source)

2) Primers

3) DNAPol

4) Buffer (8.4 pH ideal, Mg)

5) dNTPs (deoxy-ATP, CTP, GTP, TTP)

Mix everything in one tube

What are the features and requirements of the DNA polymerase used in PCR? (slide 4)

-Heat resistant polymerase

-Longevity, retain activity over long period of time

-From thermophilic bacteria (Thermus aquaticus Taq polymerase)

-Taq pol produces PCR products with a single 3' adenine overhang

Describe the three steps in the PCR reaction (slide 5)

1) Heat to separate strands

-Rapid cycling of temperature 95C to melt template strands apart, form ssDNA

2) Hybridization of Primers

-Intermediate temperatures to allow primer to anneal (hydrogen bond with template DNA)

3) DNA Synthesis from Primers/Extension

-Extension temperature (68-72C) optimum temperature to enable primers extension from 3' end by DNA polymerase

Describe what happens in the elongation step during PCR (see video)

-Polymerase binds to 3' end of primer that was annealed

-Using template strand of ssDNA, enzyme adds complementary nucleotides one by one (5' -> 3' synthesizing DNA) complementary to template DNA

-End Product: Two dsDNA molecules are formed from a single one, doubling amount of target DNA

Describe the components of the DNA sequencing reaction (slide 8)

1) dNTPs are nucleotides added to 3' end by DNApol

2) ddNTPs prevent further elongation

3) DNA Polymerase

4) DNA Primers are complementary to template, bind to template

5) DNA Template

Describe how DNA sequencing products are visualized and interpreted (slide 9 and 10)

Sanger:

-Uses 4 reaction tubes with dNTPs and separate ddNTP

-Primer labelled with radionucleotide

-Run in PCR machine

-Separate products on acrylamide gel, differing in size by 1 nucleotide

Automated Sequencing:

-Primer not radiolabelled

-ddNTPs are labelled with different chromophores

-Reaction in one tube

-Products separated on acrylamide gel

-Detector at bottom of gel, detects products by fluorescence of each band

-Trace: 5' to 3'

What components are required for copying DNA in vitro?

1) Template DNA from any source

2) Primers

What are primers?

-Short sequences of ssDNA

-Match the template DNA

-Generally 20-30 nucleotides long

True or False: Primers bind complementary sequences in single stranded template

True

What strand do forward primers copy?

What strand do reverse primers copy?

Forward Primers: Coding strand

Reverse Primers: Template strand

Here is a Coding strand.

5' G-A-T-T-C-G-T-A-C-T 3'

What will the Forward primers say?

5' G-A-T-T-C-G-T-A-C-T 3'

Here is a Template strand.

3' C-G-G-A-T-A-G-T-A-C 5'

What will the Reverse primers say?

3' C-G-G-A-T-A-G-T-A-C 5'

True or False: Primers are a match of the strand and copy the same nucleotides in the same order and direction

True

What component for copying DNA in vitro is this?

-DNA Polymerase

-Heat resistant polymerase

-Longevity, retains activity over a long period of time

Name an example of a thermophilic bacteria that is used as a DNApol

TAQ:

-Thermus aquaticus polymerase

-PCR products will have single 3' adenine overhang

What is a characteristic of PCR products made by Taq DNApol?

Single 3' adenine overhang

What buffer pH is necessary for copying DNA in vitro?

pH of 8.4

What cation is required by the buffer for enzyme activity during DNA copying in vitro?

Magnesium (Mg2+)

What is this?

-Nucleoside triphosphates

-dNTPs

-Deoxy ATP, CTP< GTP, and TTP

True or False: When copying DNA in vitro, everything is mixed in separate tubes

False, for PCR they are mixed in one tube altogether

Describe 3 steps of PCR

1) Heat to separate strands

2) Hybridization/annealing of primers

3) DNA synthesis from primers

Where do you incubate the PCR reaction?

-Thermocycler

-Allows rapid cycling of temperatures

What happens at High (95C), Intermediate and Low (68-72C) Temperatures during PCR?

High Temperatures (95°C)

-Melt template strands apart to form ssDNA

Intermediate Temperatures:

-Allow primers to anneal

-Hydrogen bond with template DNA

-Extension Temperature (68-72°C)

-Enable extension of primers from the 3' end by DNApol

What type of bonds do primers anneal with during Intermediate temperatures of PCR?

Hydrogen bond with template DNA

What end does DNApol add primers to during the Extension Temperature (68-72C)?

3' end

What happens during the First Cycle of PCR?

What is produced?

1) Region of double-stranded chromosomal DNA to be amplified

2) Separate the DNA strands and anneal primers

3) DNA synthesis

End Result: Two dsDNA molecules

What happens during the Second Cycle of PCR?

What is produced?

1) Separate the DNA strands and anneal primers

2) DNA synthesis

End Result: 2 dsDNA molecules

What happens during the Third Cycle of PCR?

What is produced?

1) Separate the DNA strands and anneal primers

2) DNA Synthesis

End Result: 8 dsDNA molecules

When does PCR stop after copying a DNA molecule?

-Reaches end of DNA

-Temperature change, switch steps of cycle

True or False: Overhang affects downstream cloning

False, it does not affect it.

You can chop the overhang away with Restriction Enzymes (REs).

How many times does PCR cycle?

30-40x cycles, produces millions of copies

Is this a dNTP or ddNTP?

-3' strand extension to hydroxyl group

-dATP, dCTP, dGTP, dTTP

-Add primers for DNA polymerase

-ssDNA molecule is sequenced

dNTP

Is this a dNTP or ddNTP?

-No hydroxyl group at 3' end, no extension when incorporated

-Small amount of dideoxyribonucleoside added by DNAPol

-Blocks further growth of DNA molecule, can't add next bp

-DNApol dissociates, you get random different sizes of DNA

ddNTP

Which DNA sequencing method is this?

-Set up four reaction tubes (dNTPs + separate ddNTP)

-Primer labelled with a radionucleotide

-Separate products on an acrylamide gel

-Chain termination method

Dideoxy Method/Sanger Sequencing

What does Acrylamide Gel do?

-Separates products differing in size by 1 nucleotide

-DNA laddering visible

Which will run faster with Dideoxy DNA Sequencing, smaller or larger DNA?

Smaller DNA will run faster and further

Which DNA sequencing method can be visualized in gel?

Dideoxy Method/Sanger Sequencing

Which DNA sequencing method is this?

-Primer is not radiolabelled

-ddNTP are each labelled with a different chromophore

-Reaction is set up in one tube

-Separated on acrylamide gel and detected via band fluorescence

Automated Sequencing

What direction is the trace of a DNA sequenced generated by Automated Sequencing?

5' to 3'

Why would GC-rich DNA be a downside when sequencing?

-Hard to denature

-DNAPol tends to skip due to repeat sequences

-Found in Neisseria gonorrhea

What does it mean if an Automated Sequence has 2 colors peaking at the same time?

Indicates SNPs (single nucleotide polymorphisms)

What is a possible cause for a machine giving false peaks during Automated Sequencing?

Long strands of adenine/guanine