Molecular bio Wk 6

What is the process of the Sanger DNA sequencing?

synthesis of one complementary strand of DNA

What are components of the Sanger DNA sequencing?

-target DNA (PCR amplicon or cloned DNA, large amounts) -1 primer (only) complementary to target region -buffer -thermostable DNA polymerase -dNTPs -ddNTPs (ratio of dNTPs : ddNTPs = 100:1)

What are dNTPs?

-2’deoxyribonucleoside triphosphates -single 3'-OH group on carbon 3 -required for chain elongation during DNA synthesis -DNA polymerases need 3'OH group

What are ddNTPs?

-2'3'-dideoxyribonucleoside triphosphates -no 3'-OH group on carbon 3 -no chain elongation possible

What are the different ddNTP colour tags?

-ddATP green -ddGTP yellow -ddCTP blue -ddTTP red

How do we analyse the results of sequencing reactions?

we need to separate out the DNA molecules according to size, measure the fluorescence given off by the incorporated ddNTPs at the 3'ends of the DNA fragments, electrophoresis (agarose, polyacrylamide)

What are the technological advances in DNA sequencing from the Human Genome Project?

-fluorescent chemistries -automation -high throughput sequencing -computational advances -increases in speed, accuracy and reductions in cost

What are repetitive sequences in the human genome?

-tandem repeats -interspersed repeats

What are tandem repeats?

-satellite DNA -minisatellites (VNTRs) -microsatellites (STRs)

What is Satellite DNA?

-large repeat units -found near centromeres and telomeres -not transcribed

What are minisatellites (VNTRs)?

-10-60 bp repeat units -found in telomeric regions -used in DNA fingerprinting

What are microsatellites (STRs)?

-1-6 bp repeat units -scattered throughout the genome -highly polymorphic -> useful in forensics and paternity tests

What are Interspersed Repeats?

-SINEs (short interspersed nuclear elements) -LINEs (long interspersed nuclear elements)

What are SINEs (Short Interspersed Nuclear Elements)?

-~100-300 bp -non-coding, transcribed but not translated

What are LINEs (Long Interspersed Nuclear Elements)?

-~6000 bp -encode reverse transcriptase -> can "copy and paste" themselves

What are SNPs (Single Nucleotide Polymorphisms)?

-a variation at a single base pair in the genome -most common type of genetic variation (occurs ~every 1,000 bases) -can be: synonymous (no change to amino acid) or non-synonymous (changes amino acid) or non-coding (can still affect gene regulation or splicing)

What are Haplotypes?

-a set of SNPs or alleles that tend to be inherited together -found close together on the same chromosome -useful in tracing inheritance, identifying disease genes

How to differentiate between SNPs and Haplotypes?

SNPs = single letter difference. Haplotype = group of SNPs

What is the basis of SNP Microarray Technologies?

-a DNA microarray contains probes for known SNP sites -sample DNA is: fragmented and labelled, hybridised to the chip, detected based on fluorescence (indicates which SNP variants are present)

What are the applications of SNP microarray technologies?

-genotyping: determines which SNP alleles a person has -GWAS (genome wide association studies): identify SNPs associated with diseases or traits -pharmacogenomics: match medications to a person's genetic profile -ancestry testing: trace lineage based on shared haplotypes