Genetics - Lecture 9: Introduction to Molecular Genotyping

Synthetic oligonucleotide primers

can be made in an automated way for replication of a target gene

Having more of which, GC or AT, increased melting point of DNA molecule? Why?

GC because it has 3 H bonds and AT only has 2

General primer guidlines

20-200 bases

~50% GC

Avoid repeats of bases

Gel electrophoresis

separates DNA fragments by size

molecules are sorted into bands on the gel by size

Dye used in agarose gel

intercalating dyes which insert themselves into DNA molecules

Ladder mixture (gel electrophoresis)

bands are compared to ladder mixture containing DNA of known sizes

Dideoxynucleotides (ddNTPs)

a nucleotide used in DNA sequencing that is missing the 3'-OH group. If a dideoxyribonucleotide is incorporated into a DNA strand, it stops any further growth of the strand.

Old school Sanger sequencing

radiolabelled primer anealled just above target sequence

four rxns, each containing primer, dNTPs, and trace levels of single ddNTPs

Four different ddNTPs

ddATP, ddCTP, ddGTP, and ddTTP

The bands on the gel in the Sanger sequencing correspond to...

one of the four different bases in a 5'---> 3' direction (top to bottom of the gel)

Each of the four Sanger reactions are run through polyacrylamide gel and read in which direction?

5'---> 3'

Modern Sanger sequencing

dNTPs + polymerase + trace amounts of flourescently labeled ddNTPs

How is modern Sanger sequencing read?

capillary electrophoresis

Why is PCR so useful?

allows us to look at one specific piece of DNA by making many copies of ONLY that piece of DNA

Requirements for PCR

-DNA template

-dNTPs

-DNA polymerase

-complimentary primers - RNA in vivo, DNA in vitro

-Mg2+ (for polymerase activity)

Step 1 PCR

heat DNA (denaturing)

Step 2 PCR

annealing of primers - both foward and reverse

Step 3 PCR

Extension-primers build new DNA (free nucleotides) in 3' to 5' direction

PCR and genotyping (indels)

Used to recognize certain mutations that give rise to different-sized PCR products (usually indels)

PCR and Sanger sequencing

The region around a known mutation can be PCR-amplified and sequenced directly

Two peaks at a specific base Sanger

heterozygous individual

RFLP

Restriction Fragment Length Polymorphism

Restriction enzyems in RFLP

recognize and cleave specific DNA sequences

What do the restriction enzymes do in RFLP?

cut the sugar-phosphate backbone at the restriction site

Mutations can remove restriction sites in a gene which...

changes the sizes and number of bands produced when the PCr product is run through the gel

Southern Blot

transfer DNA onto filter paper

add radioactively labeled probes for gene and observe autoradiography