practical 10- PCR based genotyping of polymorphisms

5 components of PCR tube

1. DNA template (original DNA to be amplified)

2. 2 single stranded primers

3. taq DNA polymerase

4. dNTPs (free nucleotides)

5. reaction buffers

3 stages of PCR

• denaturation

• annealing

• extension

denaturation

• 90-95 degrees for 30 seconds

• double DNA strand melts into open single stranded DNA

what are primers

• short (18-30 base pairs) synthetic single DNA molecules that are complementary to the beginning & end of DNA fragment that needs to be amplified

• made using software

annealing

• 50-60 degrees for 30 seconds

• drop in temperature allows primers to find their complementary sequences on single stranded DNA

• primers anneal to separated template strands in opposite directions

how to locate where primers should bind on a DNA sequence

• find the direction on DNA template strands

• primers bind antiparallel

• find complementary base sequence for primers

how to prevent template strands from re-annealing

add primers in excess so they out-compete template strand annealing

why might the temperature for annealing be different

depends on the primer sequence

extension

• primers prime synthesis of new strand by providing a short sequence of double stranded DNA

• taq polymerase extends from primers to build a new complementary strand

• it adds each dNTP to the complementary template in 5’-3’ direction

typical extension time for taq polymerase

1 min/kb

3 reasons why extension may stop

1. no more template DNA

2. no more dNTPs (unlikely)

3. temperature reaches 95 degrees again (end of the cycle)

how to work out number of DNA copies after x PCR cycles

2^{number of cycles}

what are amplicons + what cycle to they first appear

• set of double stranded amplified DNA that is the same size & is bracketed by primers

• cycle 3

how many amplicons are produced in cycle 3

2

how many amplicons are produced in cycle 4

8

what is amplicon growth like for each cycle after cycles 3/4

exponential

what is the band you see on agrose gel during gel electrophoresis + why

• amplicon

• other DNA fragments are too dilute

what are polymorphic sites

• sites in the genome where the DNA sequence in known to have individual variability

• used for genotyping

5 types of polymorphism

• SNP (single nucleotide polymorphisms): point mutations that happen from DNA replication errors → 90-95% of polymorphisms

• CNV (copy number variants): regions of our genome that vary in copy & number due to duplication or deletion → used in forensics/paternity tests

• STR (short tandem repeats): 2-7 base pairs that are repeated up to 100 times → can cause diseases

• VNTR (variable number tandem repeat): 15-200 base pairs that are repeated up to 100 times

• jumping genes (transposable elements): originate from viruses & when they are inserted into a chromosome it creates a dimorphic polymorphism → 50% of our genome

how to detect VNTR’s with gel electrophoresis

• gel has 2 alleles (bands in each lane) & bands vary in size

• typical of VNTR alleles because if sequence is repeated 10 time = 10 alleles

how to detect jumping genes with gel electrophroesis

• only 2 locations of bands in all lanes/on the gel

• alleles are 2 different sizes, one with insertion & one without

3 types of PV Alu genotypes + why

• +/+

• -/-

• +/-

• the Alu element is present at the PV 92 locus (+ allele) or absent (- allele)

3 things to mention when extracting information from gel electrophoresis

• if you can tell the allele size (control ladder/bp number)

• how many alleles (different bands)

• if patients are homozygous/hetrozygous (same/different bands in lanes)

10 pieces of equipment needed to identify the genotype of something

1. Agarose gel electrophoresis kit

2. Bench centrifuge

3. Blue and yellow tips

4. Ethidium bromide

5. Gilson Pipettes P10, P100, P1000

6. Microfuge tubes/ Eppendorfs

7. PCR tubes

8. Restriction enzyme(s)

9. Thermocycler

10. Universal tubes

4 steps for identifying a genome

1. DNA extraction from cheek cells

2. PCR

3. Digestion with restriction enzyme(s)

4. Agarose gel electrophoresis to reveal RFLP

how to calculate volume needed for PCR reaction

desired volume/stock concentration